Gene name - vein
Cytological map position - 64F1--64F5
Function - ligand for EGF receptor
Keywords - EGF-receptor gene complex
Symbol - vn
Genetic map position - 3-16.2.
Classification - EGF domain, Ig domain
Cellular location - secreted
|Recent literature||Newcomb, S., Voutev, R., Jory, A., Delker, R. K., Slattery, M. and Mann, R. S. (2018). cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg. PLoS Genet 14(8): e1007568. PubMed ID: 30142157
This study characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE many additional leg enhancers were identified, suggesting that this logic is broadly used by many genes during Drosophila limb development.
|Crossman, S. H., Streichan, S. J. and Vincent, J. P. (2018). EGFR signaling coordinates patterning with cell survival during Drosophila epidermal development. PLoS Biol 16(10): e3000027. PubMed ID: 30379844
Extensive apoptosis is often seen in patterning mutants, suggesting that tissues can detect and eliminate potentially harmful mis-specified cells. This study shows that the pattern of apoptosis in the embryonic epidermis of Drosophila is not a response to fate mis-specification but can instead be explained by the limiting availability of prosurvival signaling molecules released from locations determined by patterning information. In wild-type embryos, the segmentation cascade elicits the segmental production of several epidermal growth factor receptor (EGFR) ligands, including the transforming growth factor Spitz (TGFalpha), and the neuregulin, Vein. This leads to an undulating pattern of signaling activity, which prevents expression of the proapoptotic gene head involution defective (hid) throughout the epidermis. In segmentation mutants, where specific peaks of EGFR ligands fail to form, gaps in signaling activity appear, leading to coincident hid up-regulation and subsequent cell death. These data provide a mechanistic understanding of how cell survival, and thus appropriate tissue size, is made contingent on correct patterning.
Vein is a component of the epidermal growth factor receptor (EGF-R) pathway that is involved in signaling through the EGF receptor (EGF-R) during development of the ventrolateral epidermis and the wing. In contrast to Spitz, which must be processed to an active ligand, Vein is constitutively active. However, the activity of Vein is intrinsically weaker than that of Spitz. This is reflected by its reduced capacity to induce activated MAP kinase in cells and embryos, and the limited ability to induce ectopic expression of Egfr target genes (Schnepp, 1998 and Yarnitzky, 1998). Before discussing Vein in particular, a general word on EFG-R is in order.
The Drosophila Epidermal growth factor receptor is a ubiquitously expressed receptor tyrosine kinase. Activation of EGF-R signaling is achieved by spatially restricted extracellular factors such as ligands. For example, Drosophila Gurken is an oogenesis-specific EGF-like ligand involved in establishing egg polarity. grk transcripts are localized to the anterior-dorsal region of the oocyte and a local source of GRK is thought to activate EGF-R only in adjacent cells. Besides ligands, spatially localized cofactors can mediate the local activation of EGF-R. For example, Rhomboid and Star, which along with EGF-R are members of the spitz group responsible for structuring the ventrolateral epidermis of the embryo, modify the function of EGF-R to spatially restrict EGF-R signaling.
Vein is a candidate EGF-R ligand and is expressed in a spatially restricted manner. Vein has both an EGF domain and an immunoglobulin domain, and therefore is most closely related to the vertebrate neuregulins, which carry out their signaling function through vertebrate receptors that are related to EGF-R. The Ig-like domain of Vein, in other systems known to be involved in protein-protein interaction, may facilitate protein dimerization or interaction with other external proteins.
What role does VN play in ventrolateral signaling and in wing development? Unlike the EGF-R ligand Spitz, Vein is expressed in a localized pattern, thereby limiting Vein-EGF-R signaling to cells in the vein expression domain. In the embryo, vn and spi are expressed in overlapping domains; genetic data suggest they function together to achieve the level of EGF-R activation required for normal development of ventrolateral cells (Schnepp, 1996).
The situation is different in the wing. Vein is expressed primarily in developing interveins, while the genes that modulated Spitz/EGF-R signaling (rhomboid, Star and argos) are expressed in developing veins. EGF-R expression is down regulated in prevein cells, so that in pupal wings, EGF-R expression colocalizes with Vein in intervein regions. Therefore, it has been suggested that Spitz functions as the major ligand for activating EGF-R in vein territories, and that Vein is the major ligand for activating EGF-R in intervein regions. Spitz is broadly expressed in developing wing discs, but its active form, secreted SPI may be localized to the presumptive veins by the activity of the spitz-group members Star and Rhomboid, which are expressed in developing veins. This EGR-R signal may be activated in developing veins by SPI and may be limited to a short duration by two mechanisms: inhibition by the Spitz competitor, Argos, which is also restricted to presumptive veins, and down regulation of EGF-R transcript in the presumptive vein regions. This dampening of EGF-R signaling may be required for prevein cells to start their differentiation program (Simcox, 1996 and references).
That Vein acts to engender cell proliferation is made clear from studies of mutant vein clones. Where they appear, large vein clones reduce the size of intervein regions by an average of 28%. Effects on cell proliferation are not only locally restricted. vein mutant clones grow, but they also nonautonomously reduce the size of other compartments and of the wing as a whole. The larger the clones, the stronger the effects: a greater number of compartment boundaries and stretches of veins are affected. The wings carrying mutant clones for either vein or Rhomboid are smaller than those of control sibling flies, while the wings of double mutants are smaller still. In addition to their small size, the wings of double mutants lack all longitudinal and transverse veins. These synergistic effects indicate a close functional relationship between both vein and Rhomboid during proliferation and differentition (García-Bellido, 1994).
How do veins on the dorsal and ventral surfaces of a fly's wing line up in exact opposition to one another? The adult Drosophila wing consists of two wing surfaces, apposed by their basal membranes, which first come into contact at metamorphosis, following wing disc eversion. Contact is crucial. Veins normally appear in these surfaces in a dorsal-ventral symmetric pattern, but when contact between the two surfaces is prevented, the dorsal-ventral pattern of venation takes on a 'corrugated,' asymmetric appearance (vein cells are more compacted and more pigmented). Dorsal-ventral contact apposition was prevented during wing imaginal disc morphogenesis by implanting fragments of discs into metamorphosing hosts. In these implants, longitudinal veins differentiate in both surfaces, but exhibit wider than normal corrugation. These results and those of genetic mosaics for mutants that remove veins or cause ectopic veins, reveal mutual dorso-ventral induction/inhibition at work to modulate the final vein differentiation pattern and/or corrugation. While clones of mutants causing a lack of veins (the single mutant rhomboid, the double mutant rhomboid vein, and the triple mutant extramachrochaetaeAch rhomboid vein) are autonomous in both wing surfaces, the vein phenotype is partially rescued by wild type cells from the opposite surface. When apposed to a lack of vein differentiation in the dorsal surface, vein differentiation fails in distal vein territories, preferentially in the ventral surface. Conversely, extra differentiation as in plexus, extramachrochaetae, HS-rhomboid 27B, Notch and Delta mutants, causes extra veins in clones, not only in the wing surface of the clone, but also in the opposite wing surface. Non-autonomous effects are observed in the same wing surface, a phenomenon called 'connectivity'. Genetic mosiacs of plexus72 and emc HS-rho27B cause neighboring non-mutant cells on the same surface to differentiate extra veins, connecting them. Cis (planar) and trans (vertical) effects may be operationally related, inducing contacting cells to differentiate vein histotypes. Vein cells induce vein differentiation in neighbouring cells, either on the same surface by planar cell-cell communication, or on the opposite surface through signals along the basal membrane of the apposing epithelium. Thus, although the vein pattern is surface-autonomously generated, inhibitory (negative) and inductive (positive) signals take place between both dorsal and ventral wing surfaces in order to refine the final vein pattern with the corresponding dorso-ventral wing surfaces (Milan, 1997).
Inductive interactions between cells of distinct fates underlie the basis for morphogenesis and organogenesis across species. In the Drosophila embryo, somatic myotubes form specific interactions with their epidermal muscle attachment (EMA) cells. The establishment of these interactions is a first step toward further differentiation of the EMA cells into elongated tendon cells containing an organized array of microtubules and microfilaments. The molecular signal for terminal differentiation of tendon cells is the secreted Drosophila neuregulin-like growth factor Vein, produced by the myotubes. Although Vein mRNA is produced by all of the myotubes, Vein protein is secreted and accumulates specifically at the muscle-tendon cell junctional site. In loss-of-function vein mutant embryos, muscle-dependent differentiation of epidermal tendon cells, measured by the level of expression of specific markers (Delilah and beta1 tubulin) is blocked. When Vein is expressed in ectopic ectodermal cells, it induces the ectopic expression of these genes. These results favor the possibility that the Drosophila EGF receptor DER/Egfr expressed by the EMA cells functions as a receptor for Vein. Vein/Egfr binding activates the Ras pathway in the EMA cells leading to the transcription of the tendon-specific genes stripe, delilah, and beta1 tubulin. In Egfr1F26 mutant embryos lacking functional Egfr expression, the levels of Delilah and beta1 Tubulin are very low. The ability of ectopic Vein to induce the expression of Delilah and beta1 Tubulin depends on the presence of functional Egfrs. Activation of the Egfr signaling pathway by either ectopically secreted Spitz, or activated Ras, leads to the ectopic expression of Delilah. These results suggest that inductive interactions between myotubes and their epidermal muscle attachment cells are initiated by the binding of Vein, to the Egfr on the surface of EMA cells (Yarnitzky, 1997).
The tissue-specific regulation of Vn signaling was investigated by examining vn transcriptional control and Vn target gene activation in the embryo and the wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target genes. In the embryo, vn is a target of Spi/Egfr signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes signaling. In the larval wing pouch, vn is not a target of Spi/Egfr signaling but is expressed along the anterior-posterior boundary in response to Hedgehog (Hh) signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal-ventral boundary. The potential for vn to activate Egfr target genes correlates with its roles in development: vn has a minor role in embryogenesis and does not induce Egfr target genes such as aos and pntP1 in the embryo. Conversely, vn has a major role in wing development and Vn/Egfr signaling is a potent inducer of Egfr target genes in the wing disk. Spi also has the potential to induce Egfr target genes in the wing disk. However, the ligands appear to evoke specific responses that result in different patterns of target gene expression. Other factors modulate the potential of Vn so that induction of Vn/Egfr target genes in the wing pouch is cell specific (Wessells, 1999).
Differences between Vn and Spi are apparent in the patterns of target gene induction resultant from the ectopic expression of Vn and soluble Spitz (sSpi) in the wing pouch. Effects have been noted for three Egfr target genes: aos, pntP1, and kekkon-1 (kek1). In each case, a different response to the ligands is seen. Both ligands induce ectopic aos expression but Vn does so in a broader domain than sSpi, however, neither induces aos in the L3/L4 intervein region. In the embryo, the Egfr target gene pntP1 mediates aos induction by sSpi. Likewise in the wing, ectopic sSpi induces pntP1 expression in cells that also expressed aos. However, following ectopic Vn no detectable change in pntP1 expression is seen using in situ hybridization and only very weak induction of pntP1-lacZ is seen in a domain that does not correspond fully with ectopic aos expression. This suggests either that another transcription factor mediates the induction of aos in response to Vn or that PntP1 is capable of inducing aos, even when changes in its own expression level are too low to be detected by current methods. Both Vn and sSpi induce ectopic expression of kek1, but predominantly in posterior cells rather than throughout the domain of their ectopic expression. Thus the action of both ligands appears limited by the presence or absence of some other factor in anterior cells. There is also a difference in the level of induction: Vn, which functions to induce kek1 expression in normal development, is a potent inducer of high levels of ectopic kek1 expression, whereas sSpi induces low levels of ectopic kek1 expression and appears to reduce expression of endogenous kek1 (Wessells, 1999).
Type 1 and type 2 cDNAs differ by the inclusion of an intron between exons 4 and 5 in the processed mRNA of type II messenger, resulting in an extra 1782 bp in the type II messenger (Schnepp, 1996).
Bases in 5' UTR - 2054
Exons - 5
Bases in 3' UTR - 1131
The vein cDNAs encode two proteins that differ only in their extreme C-terminal regions. These proteins are similar to residue 606 (the end of exon 4) where they diverge because of a 1782 bp region in the larger cDNA. The type 1 protein ends with 16 amino acids that differ from the terminal 15 amino acids in the type 2 protein. There is an N-terminal signal sequence, and the protein is secreted. A PEST region is present, found in proteins with short half lives, as well as an opa (polyglutamine) region, an Ig-like domain and an EGF-like domain. There are a number of residues that are potential N-linked glycosylation sites (Schnepp, 1996).
The EGF-like domain in VN is 43 amino acids long and has the six invariant cysteines and highly conserved glycine and arginine residues characteristic of the motif. The cysteines are thought to form disulfide bonds, thereby producing a looped structure. The EGF motif in VN is between 30% and 44% identical to other EGF-like ligands. It shares 37% identity with Spitz and 33% identity with Gurken. Amino-terminal to the EGF domain is an Ig-like domain of the C2 type that includes nonimmunological proteins. The IG-like domain in VN has the two invariant cysteine residues typical of the domain. The Ig region in VN shares between 33 and 35% identity with the Ig region in the neuregulins and is 32% identical with the Ig region in NCAM (Schnepp, 1996).
date revised: 5 February 2000
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