The peak of HLHm7 expression during embryogenesis occurs at 6-10 hours. In the late blastoderm, expression is detected in a 2-3 cell-wide stripe on each side of the embryo, in groups of cells over the dorsal half of the poles, in the vitellophage, and in a dorsomedian band spanning the anterioposterior axis. During germ band extension, ectodermal expression is detected, and at the extended germ band stage, epidermal expression is abundant. In late stage 11, epidermal expression becomes patchy. At stage 10, the primordia of the supraoesophageal ganglion and the posterior midgut express HLHm7. From stage 11 through late stage 12, expression is detected in the entire mesodermal layer. From late stage 11 through stage 14, expression is also detected in the primordia of the stomatogastric nervous system and in the optic lobes (Knust, 1987).
Enhancer of split complex genes manifest distinct patterns of expression in the wing imaginal disc. m8 and m7 mRNAs are detected in clusters of cells that correspond to the locations where sensory organ precursors (SOPs) develop. In addition m8 is also detected in cells at the dorsal/ventral boundary throughout the third instar. The expression of mgamma and mdelta is at times associated with the same SOPs, and at other times, with different SOPs. However mdelta and mgamma mRNAs are only detected in a subset of proneural clusters. Like m8, mgamma is also present at high levels at the dorsal/ventral boundary in early stages. The domain of mß is the most distinctive. It is expressed in the wing blade associated with developing veins, and is also present at the dorsal/ventral boundary and wing margin, and is expressed in a complex pattern elsewhere in the disc, with no simple association with developing sensory organs (de Celis, 1996). In the eye disc, m8 and m7 are expressed spanning the morphogenetic furrow, whereas mgamma and mdelta are expressed just posterior to the furrow. mgamma, mdelta and mß are expressed in the more posterior portions of the disc, where the recruitment of undifferentiated cells into ommatidial units occurs; there is little expression of m8 and m7 in this region (de Celis, 1996).
A common consequence of Notch signaling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. Each individual E(spl) gene was expressed using the GAL4-UAS system in order to analyse each gene's effect in a number of cell fate decisions taking place in the wing imaginal disk. A focus was placed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way: they antagonize neural precursor and vein fates. Yet the efficacy of this antagonism is quite distinct: E(spl)mbeta, which is normally expressed in intervein regions, has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. While E(spl)m8 is more effective in abolishing the notum microchaeta fate, E(spl)m7 is most active against wing margin bristles (Ligoxygakis, 1999).
Drosophila neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Do E(spl) proteins function as promoter-bound repressors? The answer was sought by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. The activator domain is derived from VP16. m7Act contains the bHLH and adjacent putative helical domains from m7 but lacks the last 43 amino acids of the protein, including the C-terminal WRPW tetrapeptide required for repressor activity of the related Hairy protein. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Persistent expression of wild-type E(spl) proteins causes loss of neural precursors and sensory bristles and also suppresses wing vein formation. By contrast, m7Act efficiently induces supernumerary external sense organs, as predicted if E(spl) proteins function as direct repressors. An equivalent m7 truncation lacking the VP16 activation domain has no phenotypic effects, indicating that m7Act does not function by passively interfering with endogenous E(spl) activity, but instead acts as a transcriptional activatior. Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. These results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway (Jiménez, 1997).
echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development. Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling. Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions. Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Spencer, 2003 and references therein; Islam, 2003 and references therein).
Complicating the picture even further is an analysis of a paralogue of Ed termed friend of echinoid (fred). ed and fred transcription units are adjacent to one another, approximately 100 kilobases apart on chromosome arm 2L, but they are divergently transcribed in opposite directions. Fred acts in close concert with the Notch signaling pathway. Suppression of fred function results in specification of ectopic SOPs in the wing disc and a rough eye phenotype. Overexpression of N, Su(H), and E(spl)m7 suppresses the fred RNAi phenotypes. Accordingly, decreasing Su(H) or overexpression of Hairless enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, shows close genetic interaction with the Notch signaling pathway. The weak genetic interaction observed between fred and components of the Egfr pathway also links fred to the Egfr pathway; however, analysis of additional components of the Egfr pathway are necessary to determine Fred's role in the Egfr signaling (Chandra, 2003).
In order to study the function of fred, the heritable and inducible double-stranded RNA-mediated interference (RNAi) method was used. For this study, transcript sequence of fred was cloned as a dyad symmetric molecule in the pUAST vector and transgenic lines established. Expression of the construct was induced by crossing the transgenic lines to tissue- and/or stage-specific GAL4 driver lines. Transcription of a dyad symmetric molecule results in a RNA that snaps back to give rise to a dsRNA with a hairpin loop; this mediates the degradation of the corresponding endogenous mRNA. A 638-bp region of fred was selected for this analysis based on minimal similarity to ed sequence (Chandra, 2003).
The Notch signaling pathway is involved in limiting the SOP fate to a single cell within each proneural cluster. Since degradation of fred mRNA leads to formation of ectopic SOPs, it was of interest to see if the Notch signaling pathway genes functionally interact with fred in this process and, thus, may modulate the fred RNAi phenotype. To this end, four Notch pathway genes, Notch (N), Suppressor of Hairless [Su(H)], Hairless (H), and E (spl) m7 were tested for genetic interactions with fred (Chandra, 2003).
Among the best characterized targets of Notch signaling in Drosophila are the seven Enhancer of split [E (spl)] complex genes. Activation of the Notch signaling pathway results in the activation of the expression of various E(spl) complex genes. Overexpression of E (spl)m8, E (spl)m7, E (spl)mß, E (spl)mgamma, E (spl)m 3, and E (spl)mDelta in wing discs results in loss of sensory organs. To determine whether the phenotype associated with suppression of fred could be modulated by expression of an E(spl) complex gene, E (spl) m7 was expressed simultaneously with fred dsRNA using the pnr-GAL4 driver. Flies that overexpress both fred dsRNA and E (spl) m7 are indistinguishable from those expressing only E (spl) m7. Third instar larval wing discs from these crosses were also analyzed for A101-lacZ expression. Ectopic expression of E(spl)m7 by pnr-GAL4 results in the loss of dorsocentral and scutellar SOPs, while suppression of fred activity results in large domains of A101-positive cells. Notably, wing discs of UAS-E(spl)m7; UAS-fred RNAi/pnr-GAL4: A101-lacZ larvae show the same SOP pattern as UAS-E(spl) m7; pnr-GAL4: A101-lacZ larvae. Therefore, ectopic expression of E (spl)m7 suppresses the phenotype associated with the reduction of fred in the wing disc (Chandra, 2003).
Whether this is also the case in the developing eye was also tested. Degradation of fred mRNA in the eye with GMR-GAL4 results in a rough eye phenotype with missing or duplicated bristles and fused ommatidia. Ectopic expression of E (spl) m7 by GMR-GAL4 results in the loss of most of the bristles in the eye. While the ommatidia remain highly organized, bristle sockets are present only infrequently or are entirely missing. If present, sockets are mispositioned and sometimes duplicated. The phenotype of eyes of animals expressing both E (spl) m7 and fred ds RNA under the control of GMR-GAL4 is very similar to the phenotype of UAS-E(spl)m7/GMR-GAL4 flies, with the exception of a few fused ommatidia that can still be observed in the posterior part of the eye (Chandra, 2003).
The observations that changes in the activity of four genes of the Notch signaling pathway can either suppress or enhance the phenotypes associated with the suppression of fred function suggest that fred is functioning in close concert with the Notch signaling pathway. Reduction in the activity of a Notch signaling pathway gene, Su(H) results in an enhancement of the fred RNAi phenotype. In contrast, ectopic expression of Notch signaling pathway genes, Notch, Su(H), and E(spl)m7 suppresses, to different degrees, different aspects of the fred RNAi phenotype in the developing wing, thorax, and eye. In contrast, overexpression of Hairless (a negative regulator of the Notch pathway) enhances the phenotypes induced by Fred suppression. It is presently not clear whether Fred defines a separate pathway for SOP determination or if it shares downstream components of the Notch signaling pathway. The remarkable degree to which ectopic expression of an E(spl) complex bHLH transcription factor results in a nearly complete suppression of phenotypes associated with fred degradation strongly supports the idea of very close functional interactions. These observations, furthermore, raise the possibility that E(spl) complex genes and/or other genes of the Notch signaling pathway act downstream of fred function (Chandra, 2003).
During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, Ed has been shown to have a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed it has been shown that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. This work establishes a role for Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway (Ahmed, 2003).
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date revised: 20 March 2007
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