Emc, a Negative HLH Regulator with Multiple Functions in Drosophila Development

Emc, a negative HLH regulator with multiple functions in Drosophila development

Sonsoles Campuzano*,1

1Centro de BiologõÂa Molecular Severo Ochoa, Cantoblanco, 28049 Madrid, Spain

Expression and functional analyses of Emc have which by their own do not alter the pattern of SOs, a demonstrated that it is a prototype for a protein required recessive mutation in a gene encoding a repressor of for multiple processes in development. Initially char- AS-C would become dominant and promote the acterized as a negative regulator of sensory organ generation of extra SOs. That is, only one copy of development, it was later found to regulate many other that gene would not produce enough protein to repress developmental processes and cell proliferation. Its ability several copies of AS-C and allow a normal pattern of to block the function of bHLH proteins by forming SOs. Mutations that ful®lled this criterion were found heterodimers, which are ineective in DNA binding, in two loci: hairy (h) and the novel emc (Botas et al., accounts for the role of Emc in preventing the acquisition 1982). It was further observed that loss of emc of several cell fates which are under the control of bHLH function, in a wild type background for the AS-C, proteins. However, while maintaining this repressive caused development of extra SOs, whereas AS-C molecular mechanism, emc also appears to act as a de®ciencies suppressed this phenotype (Moscoso del positive regulator of dierentiation. Oncogene (2001) Prado and GarcõÂ a-Bellido, 1984a,b). Taken together, 20, 8299 ± 8307 these results suggested that the generation of extra SOs in emc mutants depended on the activity of the AS-C Keywords: Drosophila; extramacrochaetae; HLH; tran- and not on the modi®cation of a parallel develop- scription factor; development mental pathway. The molecular analysis of AS-C and emc con®rmed this hypothesis and provided a molecular basis for the negative regulation of AS-C Identi®cation of extramacrochaetae by emc.

According to the usual nomenclature system of Drosophila genes, the extramacrochaetae (emc) gene Emc titrates away proneural proteins was named after the ®rst discovered phenotype associated to its loss-of-function mutations, i.e., the The achaete (ac)andscute (sc) genes encode proteins development of extra sensory organs (SOs) on the which contain the basic ± helix ± loop ± helix domain adult ¯y. The larval and adult epidermis of Drosophila (bHLH) characteristic of a family of transcriptional contains thousands of SOs that are distributed regulators (Murre et al., 1989a; Villares and Cabrera, according to a stereotyped pattern. Each SO is formed 1987; see Massari and Murre, 2000, for a recent by the progeny of a single cell, the sensory mother cell review). Biochemical analysis of mammalian bHLH (SMC), which, during the third larval instar or early proteins involved in myogenesis, the MyoD family, and pupal stages, is singled out from the population of of immunoglobulin enhancer binding bHLH proteins imaginal discs cells or abdominal histoblasts (Bate, E12 and E47, showed that the HLH domain allows 1978; Bodmer et al., 1989; GarcõÂ a-Bellido and these proteins to form homodimers or heterodimers Merriam, 1971; Hartenstein and Posakony, 1989). with other members of the family, while the basic Genetic analysis of loss and gain-of-function mutations region is required for binding to speci®c DNA in the achaete-scute complex (AS-C) indicated that it sequences (Murre et al., 1989b). ac and sc are was involved in the commitment of epidermal cells to expressed at the right place and time to promote dierentiate as SOs and that the AS-C might be under SMC determination, as anticipated by the genetic some type of negative regulation (GarcõÂ a-Bellido and analyses. Both genes are coexpressed in the wing SantamarõÂ a 1978; GarcõÂ a-Bellido, 1979). Back in the imaginal discs, the anlagen of most of the thorax and early eighties, a genetic screen was performed to the wings, in clusters of cells located at precisely identify genes that might negatively regulate the AS- determined positions (Cubas et al., 1991; Romani et C (Botas et al., 1982). The rationale of the screening al., 1989; Skeath and Carroll, 1991) (Figure 1a). SMCs was that in the presence of extra doses of the AS-C, are born within these clusters of ac ± sc expressing cells (Cubas et al., 1991; Skeath and Carroll, 1991) (Figure 1a). Moreover, removal of speci®c proneural clusters in *Correspondence: S Campuzano; E-mail: [email protected] ac or sc mutants removes the corresponding SMCs and Multiples requirements for Emc in development S Campuzano 8300

Figure 1 Relationship between the pattern of expression of sc (a) and emc (b, c) in the imaginal wing disc and the pattern of SMCs. (a) Imaginal wing disc from an enhancer trap line that expresses lacZ (green) in the SMCs. The distribution of sc mRNA, revealed by in situ hybridization, is shown in purple. Note that SMCs are born within the clusters of sc expressing cells. (b) Pattern of expression of emc. It is fairly complementary to that of sc. Note minima of emc expression at the regions where the SMCs for the DC and SC bristles develop. (c) Expression of emc (purple) and distribution of SMCs (green) in a Hairy wing imaginal wing disc. (Only the prospective wing region is shown). Although sc is ubiquitously expressed in Hairy wing discs (Balcells et al., 1988), expression of emc is not modi®ed (compare with b). Note the development of ectopic SMCs at the posterior wing margin (PWM) that arise within a region of minimal emc expression. Positions of some SMCs is indicated, namely those that will give rise to the anterior notopleural bristle (ANP); anterior postalar bristle (APA); anterior wing margin bristles (AWM); dorsocentral bristles (DC); giant sensilum of the radius (GSR); sensilla of the dorsal radius (Rd); scutellar bristles (SC) and sensilla of the tegula (Td). PWB, posterior wing blade; v3, vein 3

SOs (Cubas et al., 1991; Romani et al., 1989; Skeath development of extra SOs in ectopic positions (Balcells and Carroll, 1991) and a generalized distribution of et al., 1988; Skeath and Carroll, 1991). Since AS-C Ac ± Sc products (in Hairy wing mutants) promotes proteins confer on cells the ability to become neural

Oncogene Multiples requirements for Emc in development S Campuzano 8301 precursors, the AS-C genes have been called proneural genes and the clusters of cells expressing them are referred to as the proneural clusters (Ghysen and Dambly-ChaudieÁ re, 1989; Romani et al., 1989). Another bHLH protein Daughterless (Da), previously identi®ed by its role in sex determination (Cline, 1989) acts synergistically with Ac/Sc to build the SO pattern (Caudy et al., 1988a,b; Dambly-ChaudieÁ re et al., 1988). HLH proteins have been classi®ed according to tissue distribution, dimerization capabilities and DNA-binding site speci®city (Massari and Murre, 2000). Due to their restricted pattern of expression, Ac and Sc belong to HLH class II. On the contrary, Da belongs to class I HLH proteins, which show a generalized distribution (Massari and Murre, 2000). Usually, class II HLH proteins activate transcription in the form of heterodimers with class I HLH proteins (Massari and Murre, 2000). Thus, the molecular structure of As ± Sc and Da as well as their genetic interactions (Caudy et al., 1988a,b; Dambly-ChaudieÁ re et al., 1988; Villares and Cabrera, 1987) indicated that they are transcriptional regulators that, in combination, control the activity of genes involved in SMC commitment. In agreement with this hypothesis, AS-C proteins and Da can form heterodimers that bind to speci®c DNA sites in vitro (Cabrera et al., 1987; Murre et al., 1989b). emc also encodes a transcription factor of the HLH family, but the Emc protein lacks the basic region that is involved in the interaction with DNA (Ellis et al., 1990; Garrell and Modolell, 1990) and thus, it belongs to class V of HLH proteins (Massari and Murre, 2000). Heterodimers formed by a bHLH protein and an HLH protein devoid of the basic region are unable to bind to DNA (Davis et al., 1990; Voronova and Baltimore, 1990). Since it was already demonstrated that proneural proteins speci®cally bind to DNA in the form of heterodimers (Murre et al., 1989b), a plausible hypothesis for the regulatory interactions between Emc and AS-C was put forward (Figure 2a). Emc would antagonize proneural ac ± sc function by sequestering proneural bHLH proteins in complexes incap- Figure 2 Possible mechanisms for Emc action. (a) Depicts the situation in an SMC. Sc and Da form heterodimers that are able able of eective interaction with DNA, rather than by to bind to DNA. The black rectangles indicate that the binding a direct repression of AS-C transcription (Ellis et al., domain of this heterodimer ®ts into the binding site present in the 1990; Garrell and Modolell, 1990, reviewed in Garrell promoter of the target gene. Sc/Emc and Da/Emc heterodimers and Campuzano, 1991). This mechanism would are unable to bind to DNA (indicated by the white rectangle). Commitment to the SMC fate takes place in cells like that shown account for the dose dependent interactions between in (a) in which the concentration of active heterodimers exceeds emc, as, sc and da, which suggested that a ®nely tuned that of ineective heterodimers, thus allowing activation of target balance of proneural proteins and of Emc was required gene promoter. In this case Emc plays a repressive role. A similar for proper SMC determination. It also explained how mechanism applies to the activation of Sxl in females. (b and c) the gain-of-function emc mutation Achaetous (Ach) represent two possible mechanisms for Emc to function as an activator of certain processes. (b) Represents a mesodermal cell in caused loss of SOs (GarcõÂ a-Alonso and GarcõÂ a-Bellido, which Twist (twi) homodimers activate transcription of myogenic 1988): in Ach imaginal discs there is a higher than genes, while Twist/Da heterodimers, although able to bind to normal accumulation of emc mRNA and, presumably, other promoters as indicated by the hatched rectangle, do not of Emc protein (Cubas and Modolell, 1992). This active such genes. The presence of Emc may be required for somatic muscle development to titrate away Da, allowing the mechanism for EMC action was further supported by formation of Twist homodimers. (c) A hypothetical situation in the molecular analysis of mouse myogenesis. Emc which only A/B heterodimers are able to activate the target gene shows a high degree of similarity to the murine family promoter. The ability of the C protein to sequester B in the form of Inhibitor of Dierentiation (Id) proteins (Benezra et of heterodimers unable to bind to this promoter, is counteracted al., 1990; Ellis et al., 1990; Garrell and Modolell, 1990, by the interaction of Emc with C. Note that a similar mechanism may operate in case the productive interaction between (a and b) reviewed in Norton, 2000). Like EMC, IDs are HLH takes lace after promoter binding. See text for more details about proteins that lack the basic domain (Benezra et al., (a and b)

Oncogene Multiples requirements for Emc in development S Campuzano 8302 1990) and, similarly to the AS-C regulation of Emc, Id Emc provides positional information for SO patterning negatively regulated myogenesis through the formation of non-DNA binding complexes with the myogenic The distribution of Ac/Sc in the imaginal discs bHLH factors (Benezra et al., 1990). The biochemical pre®gures the pattern of SOs, since their precursors, demonstration that Id can physically interact with the SMCs, always arise from proneural clusters (Cubas members of the bHLH family and attenuate their et al., 1991; Skeath and Carroll, 1991) (Figure 1a). binding to DNA in vitro, and that in cultured cells Id However, it has been found that generalized expression can inhibit the activation by MyoD of a structural of sc under the control of a heat shock promoter in muscle-speci®c gene enhancer, the muscle creatin ¯ies devoid of endogenous Ac and Sc promotes kinase enhancer (Benezra et al., 1990), provided strong development of bristles located at normal positions. support for the proposed mechanism of action of emc. This suggested the presence in the imaginal discs of Thus, biochemical analyses in mammalian cells and topological cues other than the patterned distribution genetic data in ¯ies converged into the discovery of a of Ac and Sc (RodrõÂ guez et al., 1990). The spatial conserved mechanism of regulation of the function of distribution of emc RNA suggested that Emc could bHLH proteins. Afterwards, it was shown that, indeed, provide some of these cues (Cubas and Modolell, 1992; EMC can form heterodimers with proteins of the AS-C Van Doren et al., 1992). In the wing imaginal disc, emc and Da and inhibit transcriptional activation of target expression is roughly complementary to that of ac/sc genes by interfering with their DNA binding ability (Figure 1b). Moreover, SMCs appear not only within (Cabrera et al., 1994; MartõÂ nez et al., 1993; Van Doren proneural clusters but also within minima of emc et al., 1991). expression, a situation that also holds for SMCs Although Emc regulates proneural protein function, developing after generalized sc expression (Cubas and it has been suggested that Emc may repress AS-C Modolell, 1992) (Figure 1c). Expression of emc is transcription indirectly since in emc mutant imaginal independent of AS-C (Cubas and Modolell, 1992). wing discs there is an increased accumulation of Sc These observations support a model in which the (Cubas et al., 1991; Cubas and Modolell, 1992). It interactions between proneural proteins and Emc re®ne has been proposed that the eect of Emc on ac ± sc the spatial distribution of cells with the ability to transcription results from its interference with ac and become SMCs. Within proneural clusters, the levels of sc self and cross-activation (MartõÂ nez et al., 1993; Emc would establish a threshold for the amount of Ac/ Van Doren et al., 1992). Considering that more Sc proteins necessary for a cell to become an SMC. recent evidence argues strongly against such cross- Only cells with levels of proneural proteins sucient to regulatory interactions occurring between the endo- titrate Emc and to activate the downstream genes of genous ac and sc genes (GoÂ mez-Skarmeta et al., the neural dierentiation pathway would be candidates 1995), alternatives have to be sought to explain the to become SMCs (reviewed in Garrell and Campuzano, deregulation of sc in emc mutants. A possibility is 1991). These cells constitute a `proneural ®eld' (Cubas the promiscuity of Id-family proteins that, not only and Modolell, 1992). Interaction among them, interfere with the function of bHLH proteins, but mediated by the Notch signaling pathway, is further can modify the DNA binding ability (either inhibiting required to restrict the number of cells that ®nally or enhancing it) of homeoproteins, ETS proteins and dierentiate as SMCs (reviewed in Artavanis-Tsakonas others, and can interfere with the activity of the Rb et al., 1999). The remaining cells of the proneural protein (reviewed in Norton, 2000). Thus, the clusters would dierentiate as epidermis. derepression of sc in emc mutants may result from Emc interfering with the regulation of sc by members of the prepattern, a postulated combination of Embryonic functions of emc transcription factors that regulate ac/sc in the imaginal disc (reviewed by Modolell and Campuzano, A current theme in development is the reiterative use of 1998). the same protein for very dierent processes. For The targeted disruption of both Id1 and Id3 genes in instance, the AS-C proteins and Da are required in mice has demonstrated that these proteins are required neurogenesis, sex determination and myogenesis (Car- for several developmental processes, including neuro- mena et al., 1995; Garrell and Campuzano, 1991; Jan genesis (Lyden et al., 1999). Expression of several and Jan, 1993). Similarly, emc is required for multiple proneural genes is expanded in such double mutants processes during Drosophila embryonic and postem- (Lyden et al., 1999). This observation is consistent with bryonic development other than the regulation of the experiments where enforced expression of Id genes pattern of bristles. Only in some cases it has been inhibited both the activity and expression of bHLH possible to relate emc requirements with the function of proneural proteins. Given the transcriptional hierar- particular bHLH proteins, suggesting that Emc may chies that operate within the proneural bHLH family interfere with the function of non-bHLH proteins. (Kageyama and Nakanishi, 1997), it seems likely that The widespread but not ubiquitous distribution of emc Ids interfere with the regulation of a given proneural transcripts in the embryo and the embryonic lethality of bHLH gene by interacting with the product of another emc null mutants (GarcõÂ a-Alonso and GarcõÂ a-Bellido, bHLH gene located further up in the hierarchy (Lyden 1988) prompted the analysis of the embryonic functions et al., 1999). of emc (Cubas et al., 1994; Ellis, 1994).

Oncogene Multiples requirements for Emc in development S Campuzano 8303 Emc is supplied maternally as shown by the invagination and the invaginating stomodeum. accumulation of emc mRNA in the ovaries and in Although emc mutant embryos do not hatch and their the syncytial blastoderm stage (Cubas et al., 1994; Ellis, cuticle displays multiple alterations (Cubas et al., 1994). This early expression corresponds to a require- 1994), no clear relationship has been established ment of Emc for sex determination. Sex is determined between the pattern of expression of emc and the emc at this stage by the transcriptional activation of the Sex mutant phenotype. lethal (Sxl) gene, which is on in females and o in Especially informative is the role of emc in the males. The ®rst zygotic transcription of Sxl depends on development of the Malpighian tubules, the excretory the X : A ratio, namely, the ratio between the number system of the ¯y, and of the trachea. Both Malpighian of X-chromosomes (X) and autosomal sets (A) (Baker, tubules and trachea are tubular structures that develop 1989) and on the action of the bHLH protein Da in two steps. In the ®rst one, there is cell proliferation (Cline, 1989). It was determined genetically that the in certain areas of the hindgut or of the ectoderm, X : A ratio is assessed by the interaction of `numerator respectively, to provide the correct number of cells that elements' present in the X-chromosomes with `denomi- will form the ®nal structure. In the second one, there nator autosomal elements' (Cline and Meyer, 1996). are cell rearrangements, which do not involve cell The X-linked genes sisterless-a (sis-a), which encodes a proliferation, but cell migration and changes of cell bZIP protein (Erickson and Cline, 1993), and sisterless- shape, which result in formation and elongation of the b,(sis-b) later identi®ed as scute, correspond to tubules (Aolter and Shilo, 2000; Skaer, 1993). In the numerator elements (Cline, 1988; Erickson and Cline, case of the Malpighian tubules, an enlarged cell, the tip 1991; Torres and SaÂ nchez, 1989, 1991). The critical role cell, appears at the distal part of each developing of Sc and Da (both bHLH proteins) in sex determina- tubule and directs and patterns cell proliferation tion suggested that denominator elements could be through activation of the Epidermal Growth Factor negative regulators of bHLH protein activity, similar Receptor (EGFR), (Baumann and Skaer, 1993). The to Emc, encoded by autosomal genes and thus present tip cell arises from a cluster of ac expressing cells and in equal amounts in males and females. Only in maintains ac expression after its singling out (Hoch et females, where Sc concentration is twice that in males, al., 1994). emc expression precedes and accompanies enough active Sc/Da heterodimers would be present to formation of the Malpighian tubules and, accordingly, overcome the inhibitory eects of the putative emc mutations drastically aect their development. autosomal-linked negative regulators and to activate Thus, in emc null mutants, tubules remain as rudiments Sxl (Figure 2a). (Da, encoded by an autosomal gene, is and contain a reduced number of cells (Cubas et al., present in equal amounts in both sexes). Indeed, 1994; Ellis, 1994). In hypomorphic emc mutants, each overexpression experiments performed in early em- tubule rudiment has several extra tip cells (Cubas et al., bryos indicated that Sxl activation depended on the 1994; Carrera et al., 1998) but these extra cells do not relative concentrations of bHLH X-linked proteins and enhance cell proliferation. Moreover, the tubules do HLH autosomal-linked proteins (Parkhurst et al., not elongate. These results suggest, ®rst, a role of emc 1990). These data were con®rmed by the identi®cation in the control of cell proliferation; second, a role in the of emc and deadpan, a class VI HLH protein, as allocation of the tip cell fate, where emc appears to denominator elements (Younger-Shepherd et al., 1992). repress ac function, and third, a role of emc in the cell Hence, the choice of male vs female depends, similar to rearrangements that lead to the elongation of the the commitment to be an SMC or an epidermoblast, tubules. on the relative concentration of bHLH and HLH With regard to tracheal development, emc is proteins (Figure 2a). expressed in cells surrounding and including the During the subsequent stages of embryogenesis, emc invaginating tracheal pits, the rudiments of the tracheal is expressed in complex patterns and, accordingly, tree (Cubas et al., 1994; Ellis, 1994). In emc mutants, several developmental processes are aected in emc the ®nal structure of the tracheal tree is often altered mutants (Cubas et al., 1994; Ellis, 1994). It is (Cubas et al., 1994). Similar phenotypes are found in noteworthy that emc mRNA is undetectable in the mutants for breathless (btl), an FGFR-type transmem- ectodermal layer from which neuroblasts segregate brane receptor required for the proper migration of the indicating that, in early embryonic neurogenesis, emc tracheal cells (Lee et al., 1996). Interestingly, Btl does not regulate proneural function. Consistently, the protein levels are reduced in emc mutants (Cubas et early patterns of proneural clusters and neural al., 1994). Moreover, expression of btl depends on the precursors appear normal in emc mutants. Note, localized expression of the bHLH-PAS transcription however, that emc is expressed at later stages in the factor trachealess (thl) (Ohshiro and Saigo, 1997). Thus developing central nervous system. Correspondingly, emc, a negative regulator of the function of bHLH before the start of germ ban retraction, emc mutants proteins, and the bHLH-PAS Thl are coexpressed in display disorganized spatial distributions of neuroblasts the tracheal pit cells and both positively regulate btl. and their descendants and, later, anomalous axonal The molecular mechanism for this positive cooperation patterns (Cubas et al., 1994). is at present unknown, but Figure 2 shows several emc mRNA accumulates in or adjacent to regions possibilities for Emc modes of action. Interestingly, that will undergo morphogenetic movements such as embryos from Id17/7 ± Id37/7 mice exhibit vascular the cephalic and ventral furrows, the amnioproctodeal malformations and the absence of branching and

Oncogene Multiples requirements for Emc in development S Campuzano 8304 sprouting of blood vessels into the neuroectoderm by HLH repressors. The eye of Drosophila comprises (Lyden et al., 1999). This suggests parallel functions of about 750 ommatidia, each composed of eight emc and Id in the formation of tubular structures. photoreceptor and several support cells. These cells Emc is also involved in the development of dierentiates in the eye imaginal disc in a stereotyped mesodermal derivatives. The mesoderm develops from order behind the so-called morphogenetic furrow (MF) the ventral-most cells of the blastoderm. These cells (Ready et al., 1976). The proneural bHLH gene atonal require Twist (Twi), a bHLH protein, for its proper (ato) is required, in combination with Da, for the speci®cation (reviewed by Baylies et al., 1998). This speci®cation of R8 (Jarman et al., 1994), the ®rst region invaginates during gastrulation and is later photoreceptor to develop and the one that subse- subdivided into somatic and visceral mesoderm which quently recruits the other photoreceptors into the give rise, respectively, to the body wall muscles developing ommatidium (Freeman, 1997). ato is ®rst (somatic muscles) and the muscles of the gut (visceral weakly expressed in a dorsoventral stripe of cells muscles) (reviewed in Baylies et al., 1998). emc is anterior and within the MF, and later is upregulated, expressed at blastoderm stage in the prospective ®rst in proneural clusters, and, ®nally, in the R8 cells mesoderm; in the mesodermal layer during gastrulation (Dokucu et al., 1996). The expression and activity of and later, in the muscle attachment sites (apodemes) ato is negatively regulated by Emc and Hairy, a bHLH (Cubas et al., 1994). Accordingly, emc mutations transcriptional repressor, which are expressed anterior profoundly aect the pattern of somatic muscles, the to the MF (Brown et al., 1995). Thus, whereas clones defects ranging from loss of some muscles to large of cells lacking either h or emc function alone have alterations of this pattern, with muscles forming large only subtle eects on photoreceptor cell development, masses detached from the epidermis (Cubas et al., combined loss of emc and h causes upregulation of ato 1994). The latter phenotype may be attributed to and the subsequent dierentiation of photoreceptor defective speci®cation of the apodemes. The function cells, in much anterior positions than in the adjacent of emc in this process may be related to that of Delilah, wild type tissue (Brown et al., 1995). These observa- a bHLH protein that is expressed almost exclusively in tions suggest that a reduction on emc and h levels is a the apodemes (Armand et al., 1994). The alteration of major prerequisite for the initiation of eye develop- the pattern of somatic muscle in emc mutants appears ment. This is precisely the case. Recently it has been to result from de®cient twi function. First, levels of shown that downregulation of h and emc is controlled Twist are decreased in emc embryos (Cubas et al., by the Notch signaling pathway, which is activated in 1994). Second, Emc may be indirectly required for Twi the eye disc cells anterior and close to the MF in function (Castanon et al., 2001) (Figure 2b). Twist can response to Hedgehog secreted by the already dier- form homodimers and heterodimers with Da in vitro entiated photoreceptors (Baonza and Freeman, 2001). (Castanon et al., 2001). Interestingly, in vivo analysis indicates that whereas Twist homodimers specify mesodermal fate, Twist-Da heterodimers repress ex- emc and vein dierentiation pression of genes required for somatic myogenesis (Castanon et al., 2001). Considering these data and the The Drosophila wing displays a stereotyped pattern of previous observation that Emc can heterodimerize with ®ve longitudinal veins and two transverse veins. This Da (Cabrera et al., 1994; MartõÂ nez et al., 1993; Van pattern is established in the imaginal wing disc by the Doren et al., 1991), during somatic muscle develop- combined action of the Notch and EGFR signaling ment Emc would be required to relieve inhibition of pathways (reviewed in de Celis, 1998). Expression of Twist by Da, by sequestering Da as Emc-Da veinlet/rhomboid (ve/rho) in prevein regions helps to heterodimers (Figure 2b). locally activate the EGFR/Ras pathway, which is emc is also expressed in the cells of the visceral instrumental to promote vein formation. EGFR/Ras mesoderm while they are spreading over the midgut activates Delta in the veins, which binds to and and indeed, cells of the visceral mesoderm showed a activates Notch at the cells ¯anking the veins. The clear migration defect in emc mutants (Cubas et al., Notch pathway in turn activates transcription of 1994). E(spl)mb, which encodes a bHLH protein that In summary, the analysis of the embryonic functions represses expression of ve/rho and prevents the of emc has shown that emc not only represses acquisition of vein fate in the cells ¯anking the pro- dierentiation but it plays positive roles, like favoring veins. The shape and position of emc clones in the wing muscle development, although mechanistically it be- and the pattern of expression of emc suggests a role for haves as a negative regulator of other proteins. this gene in vein dierentiation. Thus, clones of cells hypomorphic for emc are elongated, frequently appear to run along the veins and they also can dierentiate Emc represses the initiation of neural dierentiation in ectopic veins of normal thickness (Baonza and GarcõÂ a- the eye Bellido, 1999; Baonza et al., 2000; de Celis et al., 1995; GarcõÂ a-Alonso and GarcõÂ a-Bellido, 1988). In the pupal The initial steps of dierentiation in the Drosophila eye, wing emc is expressed at highest levels in the intervein similar to the determination of the SMCs, depend on cells ¯anking the veins (Baonza et al., 2000). These are the activity of bHLH proteins, which is counteracted the cells where Notch signaling is maximally active and

Oncogene Multiples requirements for Emc in development S Campuzano 8305 indeed, expression of emc coincides with that of Regulation of transcription and translation of Emc E(spl)mb (Baonza et al., 2000). Moreover, Baonza et al. (2000) demonstrated that Notch signaling is The above results indicate that the pattern of required from the beginning of pupal development to expression of emc and the level of accumulation of establish emc transcription pattern. This regulatory Emc protein must be ®nely controlled during develop- interaction may account for the observation that, ment. Although this is a relevant issue, not much is although the phenotype of emc clones is dierent to known of the regulation of emc. The clustering of emc that associated to loss-of-function alleles of Notch, regulatory mutations, mostly P-element insertions, in a which cause the formation of thicker veins but not the rather short stretch of DNA (Ellis et al., 1990; Garrell appearance of ectopic veins, however, when Notch and Modolell, 1990) argues against the existence of signaling is compromised, vein dierentiation becomes extended cis regulatory regions. However, the fact that very sensitive to reductions in emc function (Baonza et emc mutations associated to chromosomal breakpoints al., 2000). Moreover, both Emc and E(spl)mb map at considerable distance from the emc promoter, cooperate in the repression of ve (Baonza et al., suggests otherwise (Garrell and Modolell, 1990). 2000). Since Emc and E(spl)mb do not physically Importantly, emc transcription is not regulated by the interact (Baonza et al., 2000), Emc may titrate a AS-C (Cubas and Modolell, 1992). As indicated above, protein that inhibits the transcriptional activity of an Notch pathway regulates emc expression, although in unidenti®ed factor which co-operates with E(spl)mb in opposite ways in dierent tissues: it represses emc the repression of ve/rho (Figure 2c,d). These observa- transcription in the eye disc (Baonza and Freeman, tions and those presented above concerning eye 2001) while activates it at the prospective wing margin development, are the ®rst indications of how the region of the wing imaginal disc and at the vein/ function of an Id-family protein, Emc, is integrated intervein boundaries in pupal wings (Baonza et al., with that of an upstream signaling pathway, the Notch 2000). Although the direct eector is unknown, in the pathway, which plays a crucial and evolutionarily latter case it is known to be distinct from E(spl)mb conserved role in the control of cell determination (Baonza et al., 2000). (reviewed in Artavanis-Tsakonas et al., 1999). The polychaetoid (pyd) mutation (also called Tamou, a Japanese term for `hairy') gives rise to ectopic SOs resembling the phenotype of insuciency of emc Emc regulates cell proliferation (Chen et al., 1996; Takahisa et al., 1996). Moreover, Pyd and emc mutations act synergistically in the Cooperation of emc and the Notch pathway also development of extra veins and SOs (Takahisa et al., operates in the control of cell proliferation. In the wing 1996). pyd ectopic bristles are due to the formation of disc, cell lacking emc function do not survive but those additional SMCs (Chen et al., 1996; Takahisa et al., harboring strong hypomorphic emc alleles can pro- 1996) which appear to result from reduced transcrip- liferate, albeit to a reduced rate compared to wild type tion of emc in pyd mutant discs (Takahisa et al., cells (Baonza and GarcõÂ a-Bellido, 1999; de Celis et al., 1996). Pyd is a PDZ domain protein whose sequence 1995; GarcõÂ a-Alonso and GarcõÂ a-Bellido, 1988). Loss is closely related to mammalian Zona occludens of Notch function similarly reduces cell viability (de proteins ZO-1, ZO-2 and ZO-3, members of the Celis and GarcõÂ a-Bellido, 1994). Moreover, in the wing membrane-associated guanylate kinase homologue anlage cells doubly mutant for both emc and Notch (MAGUK) family (reviewed in Dimitratos et al., have extremely poor viability suggesting that emc and 1999). Since Pyd is associated with septate and the Notch pathway cooperate to promote cell pro- adherens junctions (Takahisa et al., 1996; Wie and liferation (Baonza et al., 2000). This interaction does Ellis, 2001), its role in emc regulation cannot be a not rely on Notch controlling emc transcription. Thus, direct one. The presence of multiple protein ± protein it has been proposed that emc and Notch signaling act interaction domains in PDZ proteins allows them to in parallel, possibly on the same set of downstream act as scaolding components to organize protein genes, to promote cell proliferation (Baonza et al., complexes (Dimitratos et al., 1999). Accordingly, it 2000). has been suggested that Pyd participates in a signaling In cultured ®broblasts, Id proteins positively control pathway that ultimately activates emc expression cell cycle progression at mid-late G1 phase by several (Takahisa et al., 1996). ways (reviewed in Norton, 2000). Namely, Id proteins Analysis of another mutation that aects the adult can relieve the inhibitory interaction of pRB with pattern of SOs, Bearded, provided indirect evidence E2F, a factor required for the transcription of S phase for a postranscriptional regulation of emc. Bearded genes. Additionally, Ids interfere with the transcrip- contains in their 3' untranslated region (UTR) several tional activation by bHLH proteins of the cyclin- copies of two heptanucleotide sequence motifs, the dependent kinase inhibitor p21WAF1/CIP1. Since me- Brd box (AGCTTTA) and the GY box (GTCTTCC). chanisms controlling cell cycle progression are evolu- Mutant Brd transcripts that are truncated in the 3' tionarily conserved, it is safe to speculate that Emc UTR due to a transposon insertion and consequently may act similarly in controlling the cell cycle in ¯ies. lack two of the three Brd boxes and the GY box, are Con®rmation of this hypothesis awaits future experi- present at elevated steady-state levels. This is con- mentation. sistent with these motifs functioning to destabilize the

Oncogene Multiples requirements for Emc in development S Campuzano 8306 wild-type transcript (Leviten et al., 1997). The Brd Perspectives box may also aect translational eciency (Lai and Posakony, 1997). emc contains in its 3' UTR a single More than 10 years have passed since the molecular Brd box and four copies of a GY box-related cloning of emc and Id, and we have gained some sequence (GTTTTCC) (Lai and Posakony, 1997). A insights on the function of emc during Drosophila role of these sequences in emc mRNA stability is development. Emc provides a clear example of how cell supported by the Achaetous mutation: This gain-of- determination relies on the cell's ability to respond to function mutation is associated with the insertion of subtle dierences in the concentration of regulatory the transposable element Tirant in the ®rst exon of proteins. However, many unresolved questions remain. emc (Garrell and Modolell, 1990) and levels of emc We do not know yet which bHLH proteins Emc does transcripts are increased in Achaetous imaginal discs interact with in many of the development processes (Cubas and Modolell, 1992). It is unknown how these aected by emc mutations. We even do not know RNA sequences regulate translation and/or mRNA whether all of these Emc-interacting proteins are stability: regulatory proteins may bind to these bHLH proteins. Such proteins can be identi®ed by sequences and regulate messenger activity or stability means of the two-hybrid system and later should be or, alternatively, formation of RNA : RNA hetero- functionally characterized by genetic analyses. Further- duplexes may regulate mRNA function (Lai and more, genetic screens can be devised to look for genes Posakony, 1998). The Emc transcript may contain that interact with emc, based for instance on the other domains susceptible of postranscriptional reg- suppression or enhancement of the adult phenotype of ulation. Thus, in vitro RNA binding experiments the emc gain-of-function mutation Achaetous. Some of demonstrate that Hel-N1, a human protein highly these approaches are currently under way and we can similar to the product of the Drosophila elav gene, can foresee the identi®cation of new Emc partners that may bind to the 3' UTR of Id mRNA. Hel-N1 binding help to shed a light into parallel processes carried out sequences are also present in the 3' UTR of the by mammalian Id proteins. Drosophila emc mRNA (King et al., 1994). Elav is an RNA binding protein known to regulate splicing of several neural speci®c genes (Lisbin et al., 2001). It Acknowledgments I would like to thank J Modolell, M Ruiz-GoÂ mezandJde would thus be interesting to determine whether Elav, Celis for comments on the manuscript. Support from which accumulates in the neurons (Robinow and DGICYT, Comunidad AutoÂ noma de Madrid and an White, 1988), plays any role in the downregulation institutional grant from FundacioÂ nRamoÂ nArecestothe of Emc which appears necessary for neural develop- Centro de BiologõÂ a Molecular Severo Ochoa is acknowl- ment to proceed. edged.

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Oncogene