BIOLOGICAL OVERVIEW

Gene activation is a wonder. It sets off an explosive division of cells, like so many magical brooms in Disney's film, "Fantasia." What is to prevent this mass of ever-dividing cells from continuing their unrestrained growth? Fortunately, gene activation is only one function of transcription factors. Another task, one that is just as important, is gene inactivation. EMC is responsible for this "negative" growth function.

extramacrochaetae encodes a transcription factor of the HLH family, but unlike other members of this family, Extramacrochaetae lacks the basic region that is involved in interaction with DNA. EMC functions as a regulator of sensory organ precursor formation by forming inactive heterodimers with proteins coded for by proneural genes. EMC also regulates formation of other organs like the midgut, suggesting a general role in regulating morphogenesis (Ellis 1994).

An example of the disruptive effects of emc mutation becomes apparent when examining the resultant nervous system. Early maturational processes appear normal, that is, up through the formation of sensory mother cells, emc mutations show no visible effects. Just prior to germ band retraction however, the spatial distribution of neuroblasts and their descendents (ganglion mother cells) become disorganized. Fasciclin II staining reveals disrupted axonal patterns. Anomalies are found in the peripheral nervous system where a disruption of neuronal groups and axonal routing has occurred.

The function of emc is required during wing morphogenesis. Mitotic recombination clones of both null and gain-of-function alleles of emc, indicate that during wing morphogenesis, emc participates in cell proliferation within the intervein regions (vein patterning), as well as in vein differentiation (de Celis, 1995). The study of relationships between emc and different genes involved in wing development reveal strong genetic interactions with genes of the Ras signaling pathway (torpedo, vein, veinlet and Gap), and with several other genes (blistered, plexus and net) in both adult wing phenotypes and cell behaviour in genetic mosaics. These interactions are also analyzed as variations of emc expression patterns in mutant backgrounds for these genes. In addition, cell proliferation behaviour of emc mutant cells varies depending on the mutant background. The results show that genes of the Ras signaling pathway are co-operatively involved in the activity of emc during cell proliferation, and later antagonistically during cell differentiation, repressing EMC expression (Baonza, 1999).

Evidence that emc acts co-operatively with genes of the Ras pathway consists of studies of wing size in flies with multiple mutations. top¹ homozygous mutant wings, mutant for the gene coding for the Epidermal growth factor receptor, are 14%-20% smaller than wild type, whereas top;emc double mutant wings are 36%-42% smaller. Surprising, the interaction between loss of function (LOF) alleles of emc and hypomorphic alleles of top and vein (vn) produces the same effect on reduction of wing size as the interaction between gain of function (GOF) hypermorphic emc and mutations in these genes. Thus, the vn wings are 15% smaller than the control wings, and in combination with the LOF and GOF alleles of emc the wings appear 27%-35% and 21%-29% smaller than wild type wings, respectively, suggesting that GOF alleles may have a LOF component (Baonza, 1999).

Evidence that emc acts antagonistically to Ras pathway genes during vein differentiation consists of observations of vein differentiation in genetic mosaics. LOF alleles of member genes of the Ras pathway exhibit the absence of veins, and conversely, mutations that cause an increase in the activity of that pathway result in the appearance of ectopic veins. Mutant vein phenotypes of LOF alleles of genes of the Ras pathway are suppressed in interactions with the LOF alleles of emc and enhanced with the GOF allele of emc. Reciprocally, the extra veins mutant phenotype of the GOF alleles for genes of the Ras pathway, is increased in interaction with LOF alleles of emc and reduced with the GOF allele of emc, indicating an antagonistic relationship between emc and genes of the Ras pathway. LOF alleles of emc rescue the lack of vein differention in emc;ve;vn triple mutant clones (ve is veinlet, coding for the protein better known as Rhomboid). Contrarily, double mutant clones of emc and alleles of members of the Ras pathway, which correspond to a hyperactivation of this pathway (Gap1 or heat shock rhomboid), differentiate ectopic veins everywhere in the wing vein (Baonza, 1999).

Genetic interactions have also shown synergistic mutant effects on venation between emc, plexus (px whose molecular nature is unknown) and net, which codes for a bHLH transcription factor. The net gene is required for intervein fate in wings. Furthermore, emc expression, which is absent in normal veins, also disappears in pupal extra veins caused by px and net. Given the molecular nature of net, the co-operative behavior wth emc could reflect direct molecular interactions. Similarly, genetic interactions and changes in expression pattern of emc are found with blistered (bs) mutants. blistered, coding for the Serum response factor of Drosophila, is expressed in the future intervein issue of the wing imaginal disc, in a complementary pattern to Ras pathway genes. In wing differentiation, bs plays a dual role in wing development. Two fully active copies of bs are required to ensure that the formation of wing veins is limited to vein territories. In addition Bs protein is essential for proper terminal differentiation of intervein cells. bs causes strong phenotypic interactions with mutants of the Ras pathway. Thus, it is proposed that emc, bs, px, net and the Ras signaling pathway set of genes are intimately related in vein/intervein patterning and differentiation. The Ras signaling pathway is thought to be involved in maintaining low levels of emc expression during vein pattern differentiation in cells that will differentiate as veins. This is consistent with observations of the expression pattern of emc. Emc protein and mRNA are found at highest levels in intervein regions (Baonza, 1999).

Some phenotypes caused by extramacrochaetae in the wing are similar to those observed when Notch signaling is compromised. Furthermore, maximal levels of extramacrochaetae expression in the wing disc are restricted to places where Notch activity is higher, suggesting that extramacrochaetae could mediate some aspects of Notch signaling during wing development. The relationships between extramacrochaetae and Notch in wing development have been studied, with emphasis on the processes of vein formation and cell proliferation. Strong genetic interaction between extramacrochaetae and different components of the Notch signaling pathway have been observed, suggesting a functional relationship between them. The higher level of extramacrochaetae expression coincides with the domain of expression of Notch and its downstream gene Enhancer of split-m beta. The expression of extramacrochaetae at the dorso/ventral boundary and in boundary cells between veins and interveins depends on Notch activity. It is proposed that at least during vein differentiation and wing margin formation, extramacrochaetae is regulated by Notch and collaborates with other Notch-downstream genes such as Enhancer of split-m beta (Baonza, 2000).

Notch mutant cells show reduced viability, whereas activation of Notch signaling causes strong mitotic activity in the wing disc, independent of the activation of vestigial and wingless. These observations suggest that Notch, in addition to its function in the establishment of the D/V boundary is also directly involved in the control of cell proliferation. In this function of Notch the genes of the E(spl) complex are not required. emc is also involved in regulating cell proliferation during wing disc development, because emc mutant cells do not proliferate at all, and clones of cells of strong emc hypomorphic alleles reduce cell proliferation in intervein territories. Mutant cells for both emc and Notch have extremely poor viability, indicating that emc and Notch cooperate to promote cell proliferation. However, this interaction does not rely on Notch controlling emc transcription, because the basal level of Emc expression in the wing pouch is not affected in Notch mutant cells. Thus, it is proposed that during imaginal cell proliferation emc and Notch signaling act in parallel, possibly on the same set of downstream genes, to promote cell proliferation (Baonza, 2000).

The activity of Notch is necessary for the formation and maintenance of the D/V boundary. Thus, loss of Notch prevents the formation of the wing margin and, conversely, ectopic Notch activity results in the formation of novel margin structures and wing outgrowths. During the third instar, Notch expression is maximal in the dorsal and ventral cells that form the D/V boundary. These cells also correspond to the places where E(spl)m beta, a Notch-downstream gene, is expressed, indicating high levels of Notch signaling there. The expression of emc at the D/V boundary is maximal in the same cells where Notch and E(spl)m beta genes are expressed, suggesting that Notch signaling could regulate emc expression. In fact, the expression of emc at the D/V border is eliminated in cells lacking Notch activity, whereas clones of cells expressing an activated form of N express ectopically high levels of Emc. Increased levels of Emc expression are also induced by the Notch ligands Dl and Ser in the dorsal and ventral compartments, respectively (Baonza, 2000).

The regulation of emc expression at the D/V boundary by Notch is not mediated by E(spl)m beta, since clones of E(spl)m beta-expressing cells do not affect the expression of emc. Elimination of E(spl)m beta or emc does not affect the formation of the wing margin, indicating that these Notch targets are not required for Notch activity in the formation of this structure. However, emc and E(spl) are required during the formation of the sensory organs characteristic of the wing margin. Thus, ectopic expression of emc [or E(spl)] throughout the wing pouch eliminates most of the sensory elements of the anterior wing margin. It is likely that this function of emc and E(spl) relies on the repression of the activity and expression of the Achaete and Scute proteins (Baonza, 2000).

The expression of several genes such as vein (ve) and blistered is restricted to either vein or intervein regions during imaginal development, indicating that at this stage the veins are being specified. A key component of vein specification is the activity of the Egfr signaling pathway, although it is not known which genes localize Egfr activation to vein territories. Both emc and Notch are required at this early stage to position vein territories and to define their extent, respectively, and it is likely that Notch and emc interact during the definition of vein territories in third instar wing discs. This interaction could be based in the regulation by Notch and Emc of similar target genes controlling the appearance and extent of vein-competent territories. However, the results suggest that in this initial establishment of vein territories the expression of emc and the activity of Notch are independent of each other, because the heterogeneity in emc expression related to developing veins observed in third instar discs is not modified in Notch mutant backgrounds. Furthermore, some characteristic phenotypes of emc clones, such as the appearance of ectopic veins of normal thickness, are never observed in Notch clones, indicating that emc and Notch are affecting independent processes during the initiation of vein development (Baonza, 2000).

After puparium formation the activity of Notch is continuously required to maintain the correct width of the vein, and at this stage Notch activation occurs in two stripes of cells adjacent to each vein. The accumulation of E(spl)m beta in these cells, as a consequence of Dl-mediated Notch activation, contributes to the restriction of ve expression to the vein, and prevents the differentiation as vein of the flanking pro-vein cells. Interestingly, the elimination of Notch or Dl activity results in the formation of thicker veins than elimination of E(spl)m beta, suggesting that additional elements are activated in response to Notch and participate in the repression of vein differentiation (Baonza, 2000).

Several arguments suggest that emc is one of these components that mediate Notch signaling during the pupal development of veins. (1) The expression of emc in pupal wings is maximal in the same cells that express E(spl)m beta, suggesting that Notch activity is responsible for the preferential accumulation of emc expression. This expression is modified when Notch activity is compromised; in such circumstances emc is detected in the novel flanking cells associated with the thickened Notch mutant veins. (2) Clones of emc mutant cells occasionally cause vein thickening, and this phenotype is greatly exaggerated in Notch and Dl mutant backgrounds, suggesting that in a situation of insufficient Notch activity, the levels of emc are critical to repress vein formation. The analysis of emc clones in l(1)N ts heterozygotes indicates that during the pupal stage cells are particularly sensitive to reduction in emc and Notch activities. In addition, clones of Dl-expressing cells induced during pupal development cause ectopic expression of emc, indicating that during this stage the activity of Notch is enough to increase the levels of emc. These results do not discard an earlier requirement for both genes in vein determination, but show that during pupal development emc and Notch do interact in the definition of vein thickness (Baonza, 2000).

The molecular basis of this interaction is unclear; so far there is no emc-target gene identified affecting vein formation. By analogy to the function of emc in antagonizing the activity of proneural proteins, it is postulated that Emc modulates the function of some protein involved in promoting vein formation. Interestingly, when both emc and E(spl)m beta are overexpressed, an enhancement of the E(spl)m beta overexpression phenotype of loss of veins is observed, suggesting that the combination of high levels of both emc and E(spl)m beta results in more effective repression of vein differentiation. Thus, it is proposed that Notch signaling, in addition to activating the expression of E(spl)m beta, induces high levels of emc expression in flanking cells, and that the combination of emc and E(spl)m beta is more efficient in suppressing vein formation than E(spl)m beta alone. Emc and E(spl) do not physically interact with each other, and therefore it is unlikely that emc contributes to E(spl)mb activity. Therefore it is suggested that Emc and E(spl)mbeta contribute to the regulation of the activity and expression of a vein-promoting protein and gene, respectively, thus explaining the observed synergy between Notch signaling and emc function in vein formation (Baonza, 2000).

GENE STRUCTURE

Bases in 5' UTR - 258

Exons - 1 of 2.7 kb

Bases in 3' UTR - 1252 bases

PROTEIN STRUCTURE

Amino Acids - 199

Structural Domains

EMC is a helix-loop-helix transcription factor lacking the basic domain that usually activates transcription (Ellis 1990 and Garrell 1990)

date revised: 20 April 98

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