The Interactive Fly

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

Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

Egfr signaling is a major regulator of ecdysone biosynthesis in the Drosophila prothoracic gland

Egfr signaling is a major regulator of ecdysone biosynthesis in the Drosophila prothoracic gland

Understanding the mechanisms that determine final body size of animals is a central question in biology. In animals with determinate growth, such as mammals or insects, the size at which the immature organism transforms into the adult defines the final body size, as adult individuals do not grow. In Drosophila, the growth period ends when the immature larva undergoes the metamorphic transition to develop the mature adult. This metamorphic transition is triggered by a sharp increase of the steroid ecdysone, synthetized in the prothoracic gland (PG), that occurs at the end of the third instar larvae (L3). It is widely accepted that ecdysone biosynthesis in Drosophila is mainly induced by the activation of tyrosine kinase (RTK) Torso by the prothoracicotropic hormone (Ptth) produced into two pairs of neurosecretory cells that project their axons onto the PG. However, the fact that neither Ptth nor torso-null mutant animals arrest larval development but only present a delay in the larva-pupa transition mandates for a reconsideration of the conventional model. This study shows that Egfr signaling, rather than Ptth/torso, is the major contributor of ecdysone biosynthesis in Drosophila. Egfr signaling was found to be activated in the PG in an autocrine mode by the EGF ligands spitz and vein, which in turn are regulated by the levels of ecdysone. This regulatory positive feedback loop ensures the production of ecdysone to trigger metamorphosis by a progressive Egfr-dependent activation of MAPK/ERK pathway, thus determining the animal final body size (Cruz, 2020).

In contrast to the developmental delay phenotype observed in larvae with reduced Ptth or torso, this study foudn that specific depletion of Drosophila homolog transducers ras(ras85D), Raf oncogene (Raf), and ERK, the core components of the MAPK/ERK pathway, in the prothoracic gland (PG) using the phmGal4 driver (phm>) induced developmental arrest at L3. This result suggests that additional RTKs might play important roles in ecdysone production. To study this possibility, all known Drosophila RTKs in the PG were knocked down and found that only depletion of Egfr phenocopied L3 arrested development observed in phm > ras85DRNAi larvae. Likewise, overexpression in the PG of a dominant-negative form of Egfr (EgfrDN) or depletion of the transcription factor pointed (pnt), the principal nuclear mediator of the Egfr signaling pathway, also resulted in arrested L3 larvae. The same results were obtained upon inactivation of Egfr or different components of the MAPK/ERK pathway using an alternative PG specific driver, amnc651Gal4. Consistent with the observed phenotypes, overexpression of a constitutively activated form of either Egfr (Egfract) or Pnt (PntP2VP16) in the PG induced premature pupariation and reduced pupal size. These results are in agreement with a previous report showing that overexpression of a constitutively activated form of Ras (RasV12) in the PG produced the same phenotype. Furthermore, overexpression of RasV12 in Egfr-depleted larvae rescued the developmental arrest phenotype and forced premature pupation. These results strongly suggest that Egfr signaling in the PG is required for the synthesis of the ecdysone pulse that triggers metamorphosis. Confirming this hypothesis, ecdysone titers in larvae depleted of either Egfr or pnt in the PG were dramatically reduced. Accordingly, Hr3 and Hr4 expression, two direct target genes of the hormone that have been used as readouts for ecdysone levels, was completely abolished in phm > EgfrRNAi and phm > pntRNAi L3 larvae compared to control animals. Moreover, addition of the active form of ecdysone, 20-hydroxyecdysone (20E), to the food rescued the developmental arrest phenotype induced by inactivation of Egfr signaling in the PG. Altogether, these results indicate that Egfr signaling in the PG endocrine cells is required for the production of the ecdysone pulse that triggers pupariation and fixes adult body size (Cruz, 2020).

Since Egfr signaling is involved in cell proliferation and survival, this study analyzed whether the above-described phenotype was due to compromised viability of PG cells. Although reduced activation of Egfr signaling diminished cell size, PG cell number and viability were not affected. Interestingly, ecdysone synthesis has been recently shown to correlate with endocycle progression and therefore cell size of PG cells. PG cells undergo three rounds of endoreplication during larval development resulting in chromatin values (C values) of 32-64 C by late L3. Remarkably, a clear reduction was observed in the C value of PG cells of phm > EgfrRNAi larvae at 120 h AEL, with most cells at 8-16 C, indicating that Egfr activation is also required to promote polyploidy in the PG cells (Cruz, 2020).

This result raised the possibility that Egfr signaling regulates ecdysone production by determining the size of the PG. To analyze this hypothesis, the effect of Egfr signaling in ecdysone production was examined. Steroidogenesis in the PG cells depends on the timely expression of ecdysone biosynthesis enzyme-encoding genes that mediate the conversion of cholesterol to ecdysone. To analyze whether Egfr signaling controls ecdysone synthesis by regulating the expression of these genes, qRT-PCR was performed in early (72 h after egg laying [AEL]), mid (96 h AEL), and late (120 h AEL) phm > EgfrRNAi and phm > pntRNAi L3 larvae. Whereas expression of the six ecdysone biosynthetic genes increased gradually from mid to late L3 in control larvae, correlating with the production of the high-level ecdysone pulse that triggers metamorphosis, inactivation of the Egfr pathway in the PG resulted in a dramatic reduction in the expression levels of neverland (nvd), spook (spo), shroud (sro), and phantom (phm) in late L3 larvae. In contrast, the expression of disembodied (dib) and shadow (sad) was not significantly reduced in Egfr-depleted larvae, which suggests that compromising Egfr signaling in the PG does not result in a general reduction in the transcriptional activity by its minor C value, as previously shown, but rather by a specific transcriptional effect. Further confirming this point, the overexpression of CycE in Egfr-depleted PGs was unable to restore normal expression of ecdysteroid biosynthetic genes nor induced proper pupariation of these animals, indicating that Egfr signaling is required for proper expression of ecdysone enzyme-encoding genes independently of promoting polyploidy of PG cells (Cruz, 2020).

As the levels of circulating ecdysone are influenced by the rates of hormone production and release, whether Egfr signaling also regulates ecdysone secretion was studied. Recently, it has been shown that ecdysone secretion from the PG cells is mediated by a vesicular regulated transport mechanism. After its synthesis, ecdysone is loaded through an ATP-binding cassette (ABC) transporter, Atet, into Syt1-positive secretory vesicles that fuse to the cytoplasmic membrane for release of the hormone in a calcium-dependent signaling. To analyze the role of Egfr signaling in this process, secretory vesicles were visualized in PG cells of phm > EgfrRNAi and phm > pntRNAi L3 larvae by expressing eGFP-tagged Syt1 (Syt-GFP) in these glands. Whereas Syt-GFP vesicles accumulate at the plasma membrane with a small number of vesicles in the cytoplasm in wild-type L3 larval PGs, a dramatic accumulation of Syt-GFP vesicles in the cytoplasm was observed in PGs with reduced Egfr signaling. Similar results were obtained when the subcellular localization of the ecdysone transporter Atet-GFP was analyzed. Consistently, overexpression of rasV12 in PGs of phm > EgfrRNAi larvae restored the subcellular localization of both Syt and Atet-GFP. Furthermore, mRNA levels of several genes involved in vesicle-mediated release of ecdysone, including Syt and Atet, were dramatically downregulated in the PG of phm > EgfrRNAi and phm > pntRNAi larvae. Therefore, the results show that Egfr signaling is also required for the vesicle-mediated release of ecdysone from PG cells. Interestingly, direct effects of Egfr signaling on the endocytic machinery have been already described in Drosophila tracheal cells as well as in human cells (Cruz, 2020).

The next question was to determine which of the EGF ligands were responsible for the Egfr pathway activation in the PG. In Drosophila, Gurken (Gur), Spitz (Spi), Keren (Krn), and Vein (Vn) serve as ligands for Egfr. Expression analysis of the four ligands revealed that only vn and spi were expressed in the PGs. Consistently, the intramembrane protease rhomboid (rho), which is necessary for the proteolytic activation of Spi, was also expressed in the PG cells. A temporal expression pattern of staged L3 PGs revealed that rho expression progressively increased during the last larval stage, while the expression of spi and vn increased sequentially, with vn upregulated at mid L3 and spi at late L3. Consistent with the expression of the ligands, mRNA levels of Egfr also showed a clear upregulation by late L3. Likewise, a specific expression of PntP2 isoform was also observed in the PG of late L3 larvae. Altogether, these results suggest that Vn and Spi might activate Egfr signaling in an autocrine manner to induce ecdysone production (Cruz, 2020).

To determine the functional relevance of each ligand, vn, spi, or both simultaneously were knocked down in the PG. As in the case of phm > EgfrRNAi, depletion of spi, vn, or both ligands at the same time caused developmental arrest in L3, although Spi appeared to have a minor effect as around 40% of phm > spiRNAi larvae underwent delayed pupariation. The attenuated effect of spi-depleted animals was probably due to a weaker effect of the spiRNAi lines as depletion of the Spi-processing protease rho in the PG resulted in all phm > rhoRNAi animals arresting development at L3. Importantly, ecdysteroid levels in mid and late L3 were significantly reduced in animals depleted of either vn or spi. Consistent with their role in controlling ecdysone production, overexpression of either Vn or an active-cleaved form of Spi in the PG induced precocious pupariation and smaller pupae. Altogether, these findings show that spi and vn act in an autocrine manner as Egfr ligands in the PG to induce ecdysone biosynthesis during the last larval stage. In fact, the correlation between vn and spi expression with the occurrence of increasing levels of ecdysteroids points to a possible positive-feedback loop regulation with 20E inducing vn and spi expression. Consistent with this possibility, vn and spi mRNA levels were reduced in PGs of ecdysteroid deficient larvae that were generated by depleting spo (phm > spoRNAi) or by overexpressing a dominant-negative form of the ecdysone receptor (phm > EcRDN). Moreover, staged PGs were cultured for 6 h ex vivo in presence or absence of 20E, and vn and spi mRNA levels were found to be significantly upregulated in the presence of the hormone. Altogether, these observations demonstrate that ecdysone exerts a positive-feedback effect on PG cells amplifying its own synthesis by inducing the expression of vn and spi. This result is consistent with a previous proposed model of ecdysone regulation in an autonomous mechanism by a positive feedback and biogenic amines. Thus, a model is proposed in which increasing levels of ecdysone promote the expression of vn and spi in the PG cells, which, in turn, increases Egfr signaling in this gland in an autocrine manner to further promote the production of ecdysone. Interestingly, it has been already shown that expression of Spi and Vn in midgut cells of Drosophila depends on ecdysone activity during metamorphosis. In addition, in vertebrates, other hormones have been postulated to control Egfr activity, such as Thyrotropin-releasing hormone, which induced the phosphorylation and activation of the Egf receptor, leading to specific transcriptional events in GH3 pituitary cells. Likewise, the Growth Hormone modulates Egfr trafficking and signaling by activating ERKs (Cruz, 2020).

Thus far, the results above show that MAPK/ERK pathway is a central regulatory element in the control of ecdysone biosynthesis in the PG, with Egfr signaling chiefly contributing to its activity. However, since Ptth/torso signaling operates through the same MAPK/ERK pathway the relative contribution of this signaling pathway in the overall activity of the PG was investigated. The fact that inactivation of Egfr signaling in the PG did not affect the mRNA expression levels of either Ptth or torso points to a minor contribution of Ptth/torso signaling in the overall MAPK/ERK activity. To analyze this possibility, the levels were compared of dpERK, a readout of MAPK/ERK activity, in PGs of phm > EgfrRNAi and phm > torsoRNAi larvae. A dramatic reduction of dpERK levels was observed in PGs of phm > EgfrRNAi larvae. Importantly, dpERK levels were also reduced in phm > torsoRNAi PGs, although to a significant lesser extent when compared to phm > EgfrRNAi larvae. Similar results were observed when nuclear accumulation of dpERK was analyzed in both larvae. Consistently, the level of activity of the MAPK/ERK pathway in phm > pntRNAi and phm > torsoRNAi larvae correlated very well with expression of the biosynthetic enzyme phm and the ecdysone-responsive genes Hr3, Hr4, and Broad-Complex (BrC), although the levels of ecdysone were significantly reduced in both cases. The different level of activation of dpERK by Egfr and Ptth/torso signaling was also consistent with the respective accumulation of Syt-GFP and Atet-GFP vesicles at the cytoplasm and the reduction of the C value of PG cells. Finally, it is important to note that the level of activity of the MAPK/ERK pathway correlated with the respective phenotypes upon inactivation of each pathway, with phm > EgfrRNAi larvae arresting development at L3 and phm > torsoRNAi larvae presenting only a delay in the pupariation time. In line with this, whereas over-activation of Egfr pathway in the PG of phm > torsoRNAi larvae induced a significant advancement in pupariation, the expression of a constitutively activated form of Torso (torsoD4021 mutants) in PGs with depleted Egfr (EgfrRNAi; torso D4021) was not able to induce precocious pupariation (Cruz, 2020).

Overall, these results show that the Egfr signaling pathway plays the main role in the biosynthesis of ecdysone by activating the MAPK/ERK pathway in the PG during mid-late L3, whereas Ptth/torso signaling acts synergistically only to increase the MAPK/ERK pathway activity thus accelerating developmental timing. In this regard, it is possible that the different strength of MAPK/ERK activation by the two signaling pathways might underline this distinct requirement of each pathway. Furthermore, temporal expression of the Egfr and Torso ligands may also contribute to the difference strengths of MAPK/ERK activation, as EGF ligands vn and spi are highly expressed during L3, whereas Ptth is only upregulated at a specific developmental time, the wandering stage. Taken together, these data suggest a model in which the increasing circulating levels of ecdysone during the last larval stage are induced by a progressive Egfr dependent activation of MAPK/ERK in the PG, whereas Ptth/torso signaling further regulates ecdysone production by integrating different environmental signals such as nutritional status, crowding conditions, and light. It is important to note that, in addition to the Egfr and Ptth/torso pathways, ecdysone biosynthesis is also regulated by the insulin/insulin-like growth factor signaling (IIS)/target of Rapamycin (TOR) signaling pathway. However, in contrast to the major role of Egfr controlling ecdysteroid levels during mid-late L3, including the strong ecdysteroid pulse that triggers pupariation, the main effect of IIS/TOR pathway is to control the production of the small ecdysteroid peak that is associated to the nutrition-dependent critical weight checkpoint that occurs at the very early L3. Thus, decreasing the IIS/TOR activity in the PG delays the critical weight checkpoint, slowing development and delaying pupariation, while increasing IIS/TOR activity in the gland induces precocious critical weight and accelerates the onset of metamorphosis. Nevertheless, it is conceivable that the increasing levels of ecdysone at the critical weight checkpoint might initiate the expression of the Egf ligands, that in turn activates the ecdysone production during mid-late L3 (Cruz, 2020).

Finally, since no role of Ptth/torso signaling has been characterized in hemimetabolous insects, it is postulated that Egfr signaling might be the ancestral ecdysone biosynthesis regulator, whereas Ptth/torso signaling has probably been co-opted in holometabolous insects during evolution to fine-tune the timing of pupariation in response to changing environmental cues. Consistent with this view, depletion of Gb-Egfr in the hemimetabolous insect Gryllus bimaculatus, where no Ptth/torso has been described, results in arrested development by the last nymphal instar. Therefore, this double regulation in holometabolous insects might provide developmental timing plasticity contributing to an appropriated adaptation to a time-limited food supply (Cruz, 2020).


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).

Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

Context-dependent changes in genetic interactions are an important feature of cellular pathways and their varying responses under different environmental conditions. However, methodological frameworks to investigate the plasticity of genetic interaction networks over time or in response to external stresses are largely lacking. To analyze the plasticity of genetic interactions, a combinatorial RNAi screen was performed in Drosophila cells at multiple time points and after pharmacological inhibition of Ras signaling activity. Using an image-based morphology assay to capture a broad range of phenotypes, the effect of 12768 pairwise RNAi perturbations was tested in six different conditions. Genetic interactions were found to form in different trajectories; an algorithm, termed MODIFI, was developed to analyze how genetic interactions rewire over time. Using this framework, more statistically significant interactions were identified compared to end-point assays, and several examples of context-dependent crosstalk between signaling pathways were further observed such as an interaction between Ras and Rel which is dependent on MEK activity (Heigwer, 2018).

proteins associated with the ras pathway


References

Cruz, J., Martin, D. and Franch-Marro, X. (2020). Egfr signaling is a major regulator of ecdysone biosynthesis in the Drosophila prothoracic gland. Curr Biol 30(8): 1547-1554. PubMed ID: 32220314

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

Heigwer, F., Scheeder, C., Miersch, T., Schmitt, B., Blass, C., Pour Jamnani, M. V. and Boutros, M. (2018). Time-resolved mapping of genetic interactions to model rewiring of signaling pathways. Elife 7. PubMed ID: 30592458

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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