Interactive Fly, Drosophila

vein interacts with components of the EGF-R signaling pathway. The most dramatic and informative of these is the resuce of vein null phenotypes by gain-of-function Epidermal growth factor receptor (Egf-r) alleles. vein null wing discs are tiny, arresting growth at the equivalent of a late second/early third-instar-size disc, however vein null discs that are heterozygous for the Egf-R Ellipse allele are rescued and grow to a large size. These discs are not fully wild-type as they have a duplicated wing pouch, which is a phenotype characteristic of vein hypomorphs. Thus the hyperactive EGF-R encoded for by the Ellipse allele can override the wing proliferation defects and partially compensate for vein loss. The Ellipse allele also rescues the larval patterning defects found in vein nulls as reflected in the phenotype of the pupal cas. These results have two major implications: (1) Egf-R appears to be epistatic to vein, which is consistent with VN acting as a ligand for EGFR. (2) A redundancy in the signaling system is suggested, whereby activation of a hyperactive receptor by another ligand(s) compensates for VN loss (Schnepp, 1996).

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the epidermal growth factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein (Martin-Bermudo, 2000).

Cell culture experiments have shown that integrins can regulate activation of the Egfr pathway at the level of the ligand, or at the level of the receptor. In the first case, integrins can regulate ligand activity through modulation of the composition and assembly of the ECM. There is increasing evidence suggesting that the binding of growth factors to the extracellular matrix is a major mechanism regulating growth factor activity. The largest group of ECM proteins that interact with growth factors include the heparan sulfates, which are the major components of the basement membrane -- indeed integrins contribute to the stabilization of the epidermal basement membrane. Integrins can also exert control on the Egfr pathway at the level of the receptor. In this scenario, the adhesion sites formed upon integrin activation (focal adhesions) can serve as recruitment points that bring together structural and signaling proteins, thus enhancing their ability to interact with the right partner, and therefore to be activated. Indeed clustering of integrins results in co-clustering of epidermal growth factor receptor molecules leading to receptor activation, and enhanced EGF-dependent activation of MAPK. In another example, integrins can also enhance the efficiency of signal transduction between the Egfr and MAPK by promoting the recruitment and activation of Raf (Martin-Bermudo, 2000).

The data presented here supports a model by which the PS integrins regulate Egfr signaling pathway at the level of its ligand Vein. This regulation involves the ability of the PS integrins to organize the tendon matrix and the basement membrane at the basal surface of muscles and tendon cells. In fact, integrin function in regulating assembly of the ECM, rather than integrin signaling, has been shown to be crucial in keratinocyte differentiation. In the absence of integrin function the levels of Vein at the muscle attachment sites are decreased compared to wild type. Therefore, it is proposed that integrins are required for the proper assembly of the basement membrane and the tendon matrix, which in turn regulates Vein activity. A role for PS2 in matrix assembly is in agreement with results showing a requirement for alpha3beta1 integrin in mediating assembly of basement membrane between the epidermis and the dermis in mice. Furthermore, results in Drosophila showing that defects in tendon cell-specific gene expression are stronger when both integrins are eliminated are consistent with data showing that the failure in assembly of the matrix is more severe in embryos lacking both PS1 and PS2 integrins than in single mutants. The basement membrane and the tendon matrix could then regulate Vein activity in different ways. (1) They could promote a higher affinity of Vein for the Egfr. In fact, heparan sulfate has been reported to promote high-affinity binding of the fibroblast growth factor 2 (FGF2) and hepatocyte growth factor (HGF) to their receptors. (2) They could also direct the movement of Vein by limiting its diffusion. This could be a mechanism for muscles to specifically transmit signals to those epidermal cells that are in contact with the same matrix, the tendon cells. (3) They could promote the accumulation or clustering of Vein to specific levels required for the activation of its receptor. And finally, (4) binding of integrins to the ECM might either protect Vein from proteolysis or lead to the production of proteolytic enzymes that release Vein from the tendon matrix and activate it. Several of these mechanisms could be operating at the same time. Thus, organization and assembly of the tendon matrix via the PS integrins would ensure the localized production and concentration of an active ligand for the Egfr at the muscle attachment sites (Martin-Bermudo, 2000).

In addition, or alternatively, the PS integrins could be required in the tendon cells to regulate Egfr function at the level of the receptor. At the muscle attachment sites of the Drosophila embryo, there are special cell junctions, called hemiadheren junctions (HAJs), which form between the ends of the muscles and the basal surface of the tendon cells in opposing pairs. HAJs are organized sites of membrane-cytoskeletal linkage which have been proposed to recruit integrins. It is worth mentioning here that although PS2 has been shown to be expressed only in the muscles, loss of PS2 integrin function affects adhesion of both muscle and epidermal HAJs. This can explain why lack of PS2 alone leads to a reduction in the expression of tendon cell-specific genes. At this level for integrin modulation of the Egfr signaling, a first step requires that epidermal HAJs act as recruitment centers for the Egfr or other signaling molecules, in the same way as focal adhesions. In this case, the detachment of the epidermal HAJs from the matrix found in embryos lacking the integrins results in the disorganization of these adhesion centers leading to a failure to cluster the Egfr and/or signaling molecules, and therefore, to activate the Egfr pathway. In this scenario it is also possible that integrins and the Egfr activate parallel pathways needed to reach the threshold level of MAPK activation, required for optimal transcription of tendon cell-specific genes (Martin-Bermudo, 2000).

This is consistent with the results presented here where over activation of the Egfr pathway can compensate for lack of integrin function. Thus, integrin-mediated cell adhesion might produce a long-lasting activation of MAPK, which cooperates with the fast and short stimulation of MAPK normally induced by activation of growth factor pathways. Experiments were performed to try to determine the relative roles of integrin adhesion versus signaling in modulating the Egfr pathway in the process of tendon cell differentiation. One of the best characterized integrin signaling events involves tyrosine phosphorylation of the focal adhesion kinase, FAK. This pathway can be mimicked by clustering the cytoplasmic domain of the betaPS subunit. It has been shown previously that clustering of the cytoplasmic tail of the bPS subunit is sufficient to initiate a signaling pathway that regulates gene expression in the Drosophila midgut. However, this signaling pathway is found to be insufficient to regulate tendon cell differentiation in the embryo. These results suggest that integrin-mediated adhesion, rather than signaling, is required to regulate tendon cell differentiation. Some experiments have shown that clustering of the cytoplasmic domain of the beta subunits does not fully mimic integrin signaling, the alpha subunits are also important and, in some cases sufficient. A pathway from integrins to MAPK has been identifed that is mediated by interactions between the transmembrane and/or extracellular domains of the alpha subunit and the adaptor protein Shc. The pathway from integrins to MAPK is alpha subunit specific, being alpha5 and alphav, which belong to the same family as the alphaPS2, the alpha subunits that signal through Shc. Therefore, it still remains possible that PS2 integrin requirements to regulate tendon cell differentiation include a signaling function through Shc (Martin-Bermudo, 2000).

TGF-alpha ligands can substitute for the neuregulin Vein in Drosophila development

ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-alpha-type and a single neuregulin-like called vein (vn). This study used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, it was found that the essential role of vn in development could be largely replaced by expression of any TGF-alpha ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. These results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity (Austin, 2014).

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