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Zygotically transcribed genes

RAS Pathway, EGF receptor-ligand complex

What is the ras pathway?

Genome-wide genetic screen identified the link between dG9a and epidermal growth factor receptor signaling pathway in vivo

In vivo severity ranking of Ras pathway mutations associated with developmental disorders

Divergent effects of intrinsically active MEK variants on developmental Ras signaling

Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut

Two Drosophilids exhibit distinct EGF pathway patterns in oogenesis


Genes of the ras pathway and Egfr receptor-ligand complex





What is the ras pathway?

The ras pathway is a signal transduction cascade. In the Drosophila eye the receptor Sevenless is borne by cells that have the potential to develop into R7 photoreceptors, the last of eight photoreceptors to differentiate in each ommatidium. Signals from the ligand Boss, a seven pass transmembrane protein that serves as the ligand for Sevenless, triggers autophosphorylation in the Sevenless receptor tyrosine kinase. Phosphorylated Sevenless binds the adaptor protein DRK which subsequently interacts with SOS, a guanine nucleotide-releasing protein, which then removes GDP from inactive RAS and substitutes GTP. The substitution of GTP for GDP activates RAS protein.

Up to this point ras pathway proteins functions not as a soup of ingredients but as an ordered complex assembled in a successive fashion to the cytoplasmic tail of the receptor Sevenless. Signal amplification is not the object, but rather assembly of a multimolecular membrane associated protein complex. Subsequent events, the activation of Draf, initiate a signal transduction cascade phosphorylating and successively activating Dmek and Rolled. This cascade does in fact serve to amplify the Sevenless signal, and results in the phosphorylation and activation of the transcription factor Pointed, which then determines R7 fate.

The ras pathway is used by four receptors, each triggered by different ligands in different tissues. The targets of Ras signaling between tissues also differ. Thus the ras pathway serves to transduce signals between receptor tyrosine kinases and the nucleus. Recent studies show that there are parallel pathways to the RAS1 to Rolled phosphorylation cascade, even in Drosophila. For a newly characterized example see Hemipterous and the dorsal closure pathway. The components of these alternative pathways are currently being investigated.

Genome-wide genetic screen identified the link between dG9a and epidermal growth factor receptor signaling pathway in vivo

G9a is one of the histone H3 Lys 9 (H3K9) specific methyltransferases first identified in mammals. Drosophila G9a (dG9a) has been reported to induce H3K9 dimethylation in vivo, and the target genes of dG9a were identified during embryonic and larval stages. Although dG9a is important for a variety of developmental processes, the link between dG9a and signaling pathways are not addressed yet.By genome-wide genetic screen, taking advantage of the rough eye phenotype of flies that over-express dG9a in eye discs, this study identified 16 genes that enhanced the rough eye phenotype induced by dG9a over-expression. These 16 genes included Star, anterior open, bereft and F-box and leucine-rich repeat protein 6 which are components of Epidermal growth factor receptor (EGFR) signaling pathway. When dG9a over-expression was combined with mutation of Star, differentiation of R7 photoreceptors in eye imaginal discs as well as cone cells and pigment cells in pupal retinae was severely inhibited. Furthermore, the dG9a over-expression reduced the activated ERK signals in eye discs. These data demonstrate a strong genetic link between dG9a and the EGFR signaling pathway (Shimazi, 2016).

In vivo severity ranking of Ras pathway mutations associated with developmental disorders

Germ-line mutations in components of the Ras/MAPK pathway result in developmental disorders called RASopathies, affecting about 1/1,000 human births. Rapid advances in genome sequencing make it possible to identify multiple disease-related mutations, but there is currently no systematic framework for translating this information into patient-specific predictions of disease progression. As a first step toward addressing this issue, a quantitative, inexpensive, and rapid framework was developed that relies on the early zebrafish embryo to assess mutational effects on a common scale. Using this assay, sixteen mutations reported in MEK1 (see Drosophila Downstream of raf1), a MAPK kinase, were assessed and a robust ranking of these mutations is provided. Mutations found in cancer were found to be are more severe than those found in both RASopathies and cancer, which, in turn, are generally more severe than those found only in RASopathies. Moreover, this rank is conserved in other zebrafish embryonic assays and Drosophila-specific embryonic and adult assays, suggesting that this ranking reflects the intrinsic property of the mutant molecule. Furthermore, this rank is predictive of the drug dose needed to correct the defects. This assay can be readily used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies (Jindal, 2017).

Divergent effects of intrinsically active MEK variants on developmental Ras signaling

Germline mutations in Ras pathway components are associated with a large class of human developmental abnormalities, known as RASopathies, that are characterized by a range of structural and functional phenotypes, including cardiac defects and neurocognitive delays. Although it is generally believed that RASopathies are caused by altered levels of pathway activation, the signaling changes in developing tissues remain largely unknown. This study used assays with spatiotemporal resolution in Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish) to quantify signaling changes caused by mutations in MAP2K1 (encoding MEK), a core component of the Ras pathway that is mutated in both RASopathies and cancers in humans. Surprisingly, it was discovered that intrinsically active MEK variants can both increase and reduce the levels of pathway activation in vivo. The sign of the effect depends on cellular context, implying that some of the emerging phenotypes in RASopathies may be caused by increased, as well as attenuated, levels of Ras signaling (Goyal, 2017).

Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut

Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, this study identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. These findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals (Xu, 2017).

This study found that the two cation channels TRPA1 and RyR are critical for cytosolic Ca2+ signaling and ISC proliferation. Under homeostatic conditions, the basal activities of TRPA1 and RyR are required for maintaining cytosolic Ca2+ in ISCs to ensure their self-renewal activities and normal tissue turnover. Agonists, including but not limited to low levels of ROS, could be responsible for the basal activities of TRPA1 and RyR. Under tissue damage conditions, increased ROS stimulates the channel activities of TRPA1 to mediate increases in cytosolic Ca2+ in ISCs. As for RyR, besides its potential to directly sense ROS, it is known to act synergistically with TRPA1 in a positive feedback mechanism to release more Ca2+ from the ER into the cytosol upon sensing the initial Ca2+ influx through TRPA1 (Xu, 2017).

Previously, Deng (2015) identified L-glutamate as a signal that can activate metabotropic glutamate receptor (mGluR) in ISCs, which in turn modulates the cytosolic Ca2+ oscillation pattern via phospholipase C (PLC) and inositol-1,4,5-trisphosphate (InsP3). Interestingly, L-glutamate and mGluR RNAi mainly affected the frequency of Ca2+ oscillation in ISCs, while their influence on cytosolic Ca2+ concentration was very weak. Strikingly, the number of mitotic cells induced by L-glutamate (i.e. an increase from a basal level of ~5 per midgut to ~10 per midgut) is far less than what has been observed in tissue damage conditions (depending on the severity of damage, the number varies from ~20 to more than 100 per midgut following damage). Consistent with this, in a screen for regulators of ISC proliferation in response to tissue damage, this study tested three RNAi lines targeting mGluR (BL25938, BL32872, and BL41668, which was used by Deng, 2015), and none blocked the damage response in ISCs, suggesting that L-glutamate and mGluR do not play a major role in damage repair of the gut epithelium (Xu, 2017).

This study found that ROS can trigger Ca2+ increases through the redox- sensitive cation channels TRPA1 and RyR under damage conditions. In particular, it was demonstrated using voltage-clamp experiments that the TRPA1-D isoform, which is expressed in the midgut, is sensitive to the oxidant agent paraquat. In addition, the results of previous studies have demonstrated the direct response of RyR to oxidants via single channel recording and showed that RyR could amplify TRPA1-mediated Ca2+ signaling through the Ca2+-induced Ca2+ release (CICR) mechanism. Interestingly, expression of oxidant- insensitive TRPA1-C isoform in the ISCs also exhibits a tendency to induce ISC proliferation, indicating that ROS may not be the only stimuli for TRPA1 and RyR under physiological conditions. Possible other activators in the midgut may be irritant chemicals, noxious thermal/mechanical stimuli, or G-protein-coupled receptors (Xu, 2017).

Altogether, the concentration of cytosolic Ca2+ in ISCs appears to be regulated by a number of mechanisms/inputs including mGluR and the ion channels TRPA1 and RyR. Although mGluR might make a moderate contribution to cytosolic Ca2+ in ISCs, TRPA1 and RyR have a much stronger influence on ISC Ca2+ levels. Thus, it appears that the extent to which different inputs affect cytosolic Ca2+ concentration correlates with the extent of ISC proliferation (Xu, 2017).

Although, as a universal intracellular signal, cytosolic Ca2+ controls a plethora of cellular processes, we were able to demonstrate that cytosolic Ca2+ levels regulate Ras/MAPK activity in ISCs. Specifically, we found that trpA1 RNAi or RyR RNAi block Ras/MAPK activation in stem cells, and that forced cytosolic Ca2+ influx by SERCA RNAi induces Ras/MAPK activity. Moreover, Ras/MAPK activation is an early event following increases in cytosolic Ca2+, since increased dpErk signal was observed in stem cells expressing SERCA RNAi before they undergo massive expansion, and when Yki RNAi was co-expressed to block proliferation. It should be noted that a more variable pattern of pErk activation was observed with prolonged increases of cytosolic Ca2+, suggesting complicated regulations via negative feedback, cross-activation, and cell communication at late stages of Ca2+ signaling. This might explain why Deng failed to detect pErk activation after 4 days induction of Ca2+ signaling (Deng, 2015). Previously, Ras/MAPK activity was reported to increase in ISCs, regulating proliferation rather than differentiation, in regenerating midguts, which is consistent with the findings about TRPA1 and RyR (Xu, 2017).

The Calcineurin A1/CREB-regulated transcription coactivator/CrebB pathway previously proposed to act downstream of mGluR-calcium signaling (Deng, 2015) is not likely to play a major role in high Ca2+-induced ISC proliferation, as multiple RNAi lines targeting CanA1 or CrebB were tested and none of them suppressed SERCA RNAi-induced ISC proliferation. In support of this model, it was also found that the active forms of CanA1/ CRTC/ CrebB cannot stimulate mitosis in ISCs when their cytosolic Ca2+ levels are restricted by trpA1 RNAi, whereas mitosis induced by the active forms of Ras or Raf is not suppressed by trpA1 RNAi (Xu, 2017).

Prior to this study, it has been shown that paracrine ligands such as Vn from the visceral muscle, and autocrine ligands such as Spi and Pvf ligands from the stem cells, can stimulate ISC proliferation via RTK-Ras/MAPK signaling. It study found that multiple RTK ligands in the midgut are down-regulated by trpA1 RNAi expression in the ISCs, including spi and pvf1 that can be induced by SERCA RNAi. Further, it was demonstrated that high Ca2+ fails to induce ISC proliferation in the absence of EGFR. As spi is induced by EGFR-Ras/MAPK signaling in Drosophila cells, and DNA binding mapping (DamID) analyses indicate that spi might be a direct target of transcriptional factors downstream of EGFR-Ras/MAPK in the ISCs, the autocrine ligand Spi might therefore act as a positive feedback mechanism for EGFR-Ras/MAPK signaling in ISCs (Xu, 2017).

In summary, this study identifies a mechanism by which ISCs sense microenvironment stress signals. The cation channels TRPA1 and RyR detect oxidative stress associated with tissue damage and mediate increases in cytosolic Ca2+ in ISCs to amplify and activate EGFR-Ras/MAPK signaling. In vertebrates, a number of cation channels, including TRPA1 and RyR, have been associated with tumor malignancy. The current findings, unraveling the relationship between redox-sensing, cytosolic Ca2+, and pro-mitosis Ras/MAPK activity in ISCs, could potentially help understand the roles of cation channels in stem cells and cancers, and inspire novel pharmacological interventions to improve stem cell activity for regeneration purposes and suppress tumorigenic growth of stem cells (Xu, 2017).

Two Drosophilids exhibit distinct EGF pathway patterns in oogenesis

Deciphering the evolution of morphological structures is a remaining challenge in the field of developmental biology. The respiratory structures of insect eggshells, called the dorsal appendages, provide an outstanding system for exploring these processes since considerable information is known about their patterning and morphogenesis in Drosophila melanogaster and dorsal appendage number and morphology vary widely across Drosophilid species. This study investigated the patterning differences that might facilitate morphogenetic differences between D. melanogaster, which produces two oar-like structures first by wrapping and then elongating the tubes via cell intercalation and cell crawling, and Scaptodrosophila lebanonensis, which produces a variable number of appendages simply by cell intercalation and crawling. Analyses of BMP pathway components thickveins and P-Mad demonstrate that anterior patterning is conserved between these species. In contrast, EGF signaling exhibits significant differences. Transcripts for the ligand encoded by gurken localize similarly in the two species, but this morphogen creates a single dorsolateral primordium in S. lebanonensis as defined by activated MAP kinase and the downstream marker broad. Expression patterns of pointed, argos, and Capicua, early steps in the EGF pathway, exhibit a heterochronic shift in S. lebanonensis relative to those seen in D. melanogaster. This study demonstrated that the S. lebanonensis Gurken homolog is active in D. melanogaster but is insufficient to alter downstream patterning responses, indicating that Gurken-EGF receptor interactions do not distinguish the two species' patterning. Altogether, these results differentiate EGF signaling patterns between species and shed light on how changes to the regulation of patterning genes may contribute to different tube-forming mechanisms (O'Hanlon, 2017).

proteins associated with the ras pathway


References

Deng, H., Gerencser, A. A. and Jasper, H. (2015). Signal integration by Ca(2+) regulates intestinal stem-cell activity. Nature 528(7581): 212-217. PubMed ID: 26633624

Goyal, Y., Jindal, G. A., Pelliccia, J. L., Yamaya, K., Yeung, E., Futran, A. S., Burdine, R. D., Schupbach, T. and Shvartsman, S. Y. (2017). Divergent effects of intrinsically active MEK variants on developmental Ras signaling. Nat Genet [Epub ahead of print]. PubMed ID: 28166211

Jindal, G. A., Goyal, Y., Yamaya, K., Futran, A. S., Kountouridis, I., Balgobin, C. A., Schupbach, T., Burdine, R. D. and Shvartsman, S. Y. (2017). In vivo severity ranking of Ras pathway mutations associated with developmental disorders. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 28049852

O'Hanlon, K. N., Dam, R. A., Archambeault, S. L. and Berg, C. A. (2017). Two Drosophilids exhibit distinct EGF pathway patterns in oogenesis. Dev Genes Evol 228(1):31-48. PubMed ID: 29264645

Shimaji, K., Konishi, T., Yoshida, H., Kimura, H. and Yamaguchi, M. (2016). Genome-wide genetic screen identified the link between dG9a and epidermal growth factor receptor signaling pathway in vivo. Exp Cell Res [Epub ahead of print]. PubMed ID: 27343629

Xu, C., Luo, J., He, L., Montell, C. and Perrimon, N. (2017). Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut. Elife 6. PubMed ID: 28561738

Zygotically transcribed genes

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