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
  • Tissue-autonomous immune response regulates stress signalling during hypertrophy
  • 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
  • The Drosophila ortholog of mammalian transcription factor Sox9 regulates intestinal homeostasis and regeneration at an appropriate level
  • ClC-c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila
  • The Yun/Prohibitin complex regulates adult Drosophila intestinal stem cell proliferation through the transcription factor E2F1
  • Sequential Ras/MAPK and PI3K/AKT/mTOR pathways recruitment drives basal extrusion in the prostate-like gland of Drosophila
  • Drosophila RASopathy models identify disease subtype differences and biomarkers of drug efficacy
  • Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants

  • 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).

    Tissue-autonomous immune response regulates stress signalling during hypertrophy

    Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via Ras(V12)-overexpression in the postmitotic salivary glands of Drosophila larvae, the glands adaptability to growth signals and induced hypertrophy was overridden. The accompanied loss of tissue integrity, recognition by cellular immunity and cell death are all buffered by blocking stress signalling through a genuine tissue-autonomous immune response. This novel, spatio-temporally tightly regulated mechanism relies on the inhibition of a feedback-loop in the JNK-pathway by the immune effector and antimicrobial peptide Drosomycin. While this interaction might allow growing salivary glands to cope with temporary stress, continuous Drosomycin expression in Ras(V12)-glands favors unrestricted hypertrophy. These findings indicate the necessity to refine therapeutic approaches that stimulate immune responses by acknowledging their possible, detrimental effects in damaged or stressed tissues (Krautz, 2020).

    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).

    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).

    The Drosophila ortholog of mammalian transcription factor Sox9 regulates intestinal homeostasis and regeneration at an appropriate level

    Balanced stem cell self-renewal and differentiation is essential for maintaining tissue homeostasis, but the underlying mechanisms are poorly understood. This study identified the transcription factor SRY-related HMG-box (Sox) 100B, which is orthologous to mammalian Sox8/9/10, as a common target and central mediator of the EGFR/Ras and JAK/STAT signaling pathways that coordinates intestinal stem cell (ISC) proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair in Drosophila. The two stress-responsive pathways directly regulate Sox100B transcription via two separate enhancers. Interestingly, an appropriate level of Sox100B is critical for its function, as its depletion inhibits ISC proliferation via cell cycle arrest, while its overexpression also inhibits ISC proliferation by directly suppressing EGFR expression and additionally promotes ISC differentiation by activating a differentiation-promoting regulatory circuitry composed of Sox100B, Sox21a, and Pdm1. Thus, this study reveals a Sox family transcription factor that functions as a stress-responsive signaling nexus that ultimately controls tissue homeostasis and regeneration (Jin, 2020).

    Homeostatic renewal of many adult tissues requires balanced stem cell proliferation and differentiation, a process that is commonly compromised in cancer and in tissue degenerative diseases. The intestinal epithelium in adult Drosophila midgut provides a genetically tractable system for understanding the underlying mechanisms of tissue homeostasis and regeneration driven by resident stem cells. The intestinal stem cells (ISCs) of the Drosophila midgut normally divide to renew themselves and give rise to two different types of progenitor cells that respectively differentiate into enterocyte cells (ECs) and enteroendocrine cells (EEs). Normally, ISCs divide occasionally and thereby maintain the ongoing renewal of the epithelium, a slow process that takes approximately 2-4 weeks. However, upon damage or infection, ISCs are able to rapidly divide to facilitate accelerated epithelial repair in as fast as two days (Jin, 2020).

    Extensive studies have implicated the JAK/STAT and the EGFR/Ras/mitogen-activated protein kinase (MAPK) as the two major signaling pathways that regulate ISC proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair. The EGFR signaling is considered to play a predominant role in the regulation of ISC proliferation because it is required for the JAK/STAT signaling activation-induced ISC proliferation, whereas the JAK/STAT signaling is not essential for EGFR/Ras signaling activation-induced ISC proliferation. The EGFR signaling is also important for remodeling of the differentiated cells, including the exclusion of damaged/aged ECs and incorporation of new cells. The JAK/STAT pathway is also essential for ISC differentiation. ISCs with compromised JAK/STAT activity generate progenitor cells that are incapable of further differentiation. Despite the importance of the two signaling pathways in controlling intestinal homeostasis, their downstream targets-which integrate pathway activities to coordinate ISC proliferation and differentiation-remain elusive (Jin, 2020).

    Sox (SRY-related HMG-box) family transcription factors (TFs) are known to have diverse roles in cell-fate specification and differentiation in multicellular organisms. In mouse-small intestine, Sox9, a SoxE subfamily member, is expressed in ISCs to regulate ISC proliferation and differentiation, but whether it acts as an oncogene or a tumor suppressor is still in debate. In Drosophila midgut, Sox21a, a SoxB2 subfamily member, is specifically expressed in ISCs and transient progenitor cells, and is essential for progenitor cell differentiation into mature cells (Chen, 2016, Zhai, 2015, Zhai, 2017). This study identified Sox100B, the Drosophila ortholog of Sox9, as a common downstream gene target for both the JAK/STAT and the EGFR signaling in regulating ISC proliferation and differentiation. This study also revealed that an appropriate level of Sox100B is critical for its function in regulating ISC proliferation, in that it may allow it to serve as an important mediator for a balanced process of ISC proliferation and differentiation, thereby maintaining intestinal homeostasis (Jin, 2020).

    Although it has been well established that in the Drosophila midgut, the stress-responsive JAK/STAT signaling and EGFR/Ras/MAPK signaling are the two major signaling pathways that regulate ISC proliferation and differentiation, the downstream signaling targets that coordinate ISC proliferation and differentiation for intestinal regeneration are still yet to be identified. Sox100B identified in this study may represent such a key target. First, the expression of Sox100B is regulated by both JAK/STAT- and EGFR-signaling pathways. Normally Sox100B is expressed specifically in ISCs and EBs, where JAK/STAT- and EGFR/Ras/MAPK-pathway activities are high, and its expression is highly dependent on the activity of JAK/STAT- and EGFR/Ras/MAPK-signaling activities. Second, similar to the functions of JAK/STAT and EGFR signaling, Sox100B is critically required for both ISC proliferation and differentiation. The sustained EGFR/Ras/MAPK activity in EBs is important for the initiation of DNA endoreplication during the process of EC differentiation, and the sustained JAK/STAT signaling activity in EBs is essential for terminal differentiation toward both EC and EE lineage. Depletion of Sox100B causes ISC quiescence, similar to that caused by the disruption of EGFR signaling, as well as arrest of EB differentiation, similar to that caused by the disruption of JAK/STAT signaling. Third, an appropriate level of Sox100B expression appears to be critical for intestinal homeostasis. This effect by the expression level, as well as its responsiveness to JAK/STAT, EGFR, and potentially other stress-induced signaling activities (not shown), such as Wnt and Hippo signaling, may position Sox100B as a central mediator that coordinates ISC proliferation and differentiation during intestinal homeostasis and regeneration in Drosophila (Jin, 2020).

    Sox100B is a Sox family group-E transcription factor, homolog of mammalian Sox8/9/10. In mouse small intestine, Sox9 is expressed in stem cells and progenitor cells at the base of crypts, and loss of Sox9 in the intestinal epithelium causes ISC hyperplasia and failure of Paneth cell differentiation (Bastide, 2007, Mori-Akiyama, 2007). Interestingly, in the stem cell zone, Sox9 is expressed at low levels in ISCs and high levels in the quiescent or reserved stem cells that are also considered as the secretory progenitors. A possible explanation for these observations is that a low level of Sox9 sustains actively dividing ISCs, while an increase of SOX9 converts these proliferating ISCs into quiescent ISCs that will eventually differentiate into Paneth cells. Similarly, Sox9 is also implicated in regulating colorectal cancer cells, but there are conflicting data regarding whether Sox9 functions as an oncogene or a tumor suppressor. These seemingly contradictory results can be reconciled with a proposed model that Sox9 functions at an appropriate level, with a critical dose of Sox9 that exhibits proliferation-promoting activity, while increasing or decreasing this dose both result in proliferation-inhibitory activity. It is worthy to note that the differentiation-promoting function of Sox9 could potentially further complicate the interpretation of the mutant phenotype. It has been shown in Drosophila gut that defects in differentiation can induce a stressed microenvironment that promotes cell proliferation and propels tumor development (Jin, 2020).

    The results of this study suggest many aspects of functional conservation of this Sox E subfamily gene in ISCs from Drosophila to mammals. Sox100B regulates both ISC proliferation and differentiation in the Drosophila intestine, and in terms of regulating ISC proliferation, Sox100B also requires an appropriate expression level. This study has demonstrated that this modulation of Sox100B expression is largely due to a negative feedback mechanism, in which increased Sox100B caused by elevated EGFR/Ras/MAPK signaling in turn suppresses the expression of EGFR, thereby leading to damped EGFR-signaling activity. Of note, contradictory data were recently reported on the roles of Sox100B and Sox21a in regulating ISC proliferation: both a proliferation-promoting role and a tumor-suppressive role for Sox21a in ISCs have been reported; as for the role of Sox100B, it has been shown in an RNAi genetic screen that Sox100B is required for P.e.-induced ISC proliferation, whereas another study showed that depletion of Sox100B by RNAi causes increased ISC proliferation. Consideration of the effects caused by different levels of Sox100B expression that was observed in the present study may help resolve understanding of apparently disparate functions for these genes as central coordinators of both ISC proliferation and differentiation. It is proposed that, normally, a low level of Sox protein expression sustains ISC proliferation. A transient increase of Sox protein may not only promote cell cycle exit but also activate programs for terminal differentiation, thereby leading to a coordinated ISC proliferation and differentiation and, consequently, a coherent process of epithelial renewal (Jin, 2020).

    This study demonstrates that Sox100B directly regulates Sox21a to promote differentiation. One important downstream target of Sox100B and Sox21a appears to be Pdm1, a known EC-fate-promoting factor. Interestingly, overexpression of Pdm1 in progenitor cells rapidly shuts down both Sox100B and Sox21a expression, indicating a negative feedback mechanism. Therefore, the induced Sox100B-Sox21a-Pdm1 axis in the differentiating ECs not only promotes cell differentiation, but also acts in a feedback mechanism to turn down EGFR and JAK/STAT signaling activities, thereby allowing ECs to terminally differentiate. This differentiation-promoting axis might also have a role in turning down ISC-specific programs, which are independently regulated by EGFR or JAK/STAT signaling pathways. For example, downregulation of the stem-cell-factor Esg is required for EB differentiation, and ectopic expression of Pdm1 is able to antagonize Esg expression in progenitor cells. These kinds of feedback regulation could be a common strategy used for initiation and finalization of a cell-differentiation program (Jin, 2020).

    In summary, this study identified the transcription factor Sox100B as a major effector downstream of JAK/STAT and EGFR pathways that acts at an appropriate level to coordinate ISC proliferation and differentiation during both normal intestinal homeostasis and during damage- and infection-induced intestinal regeneration in Drosophila. With the 'just-right' effect endowed by a feedback mechanism, Sox100B behaves as a homeostatic sensor in the intestinal epithelium that coordinates stem cell proliferation with stem cell differentiation under various environmental conditions. It is proposed that this expressional and functional modulation associated with Sox family transcription factors may be a general mechanism for maintaining tissue homeostasis and regeneration in many organs, including those in mammals, and that deregulation of this mechanism may lead to tissue degeneration or cancer development (Jin, 2020).

    ClC-c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila

    Adult stem cells uphold a delicate balance between quiescent and active states, which is crucial for tissue homeostasis. Whereas many signalling pathways that regulate epithelial stem cells have been reported, many regulators remain unidentified. This study used flies to generate tissue-specific gene knockdown and gene knockout. qRT-PCR was used to assess the relative mRNA levels. Immunofluorescence was used to determine protein localization and expression patterns. Clonal analyses were used to observe the phenotype. RNA-seq was used to screen downstream mechanisms. It is reported that a member of the chloride channel family, ClC-c, which is specifically expressed in Drosophila intestinal stem/progenitor cells and regulates intestinal stem cell (ISC) proliferation under physiological conditions and upon tissue damage. Mechanistically, it was found that the ISC loss induced by the depletion of ClC-c in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway. These findings reveal an ISC-specific function of ClC-c in regulating stem cell maintenance and proliferation, thereby providing new insights into the functional links among the chloride channel family, ISC proliferation and tissue homeostasis (Huang, 2022).

    The Yun/Prohibitin complex regulates adult Drosophila intestinal stem cell proliferation through the transcription factor E2F1

    Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. From an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), this study identified a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. It was demonstrated that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, this study found that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, this study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation (Zho, 2022).

    Prohibitins (PHBs) are members of the conserved SPFH superfamily. PHB1 was first identified by its antiproliferative activity upon ectopic expression, which was later attributed to its 3' untranslated region instead of the PHB protein itself. The PHB complex contains two homologous members: PHB1 and PHB2. PHB1 and PHB2 are ubiquitously expressed and are present in the mitochondria, the nucleus, cytosol, and the lipid rafts of the plasma membrane. A number of studies have described a role of the PHB complex within the mitochondria, where it forms a supramacromolecular structure at the inner membrane of the mitochondria acting as a scaffold (or a chaperone) for proteins and lipids regulating mitochondrial metabolism. The PHB complex has been implicated in various cellular and developmental processes and diseases, such as mitochondrial respiration, signaling, and mitophagy depending on its cellular localization. Disruption of the PHB genes has effects ranging from decreased replicative lifespan in yeast, to larval arrest in Drosophila, and to embryonic lethality in mice. However, it remains unexplored whether they play a role in intestinal stem cell regulation in Drosophila (Zho, 2022).

    Proliferation and differentiation of adult stem cells must be tightly controlled to maintain tissue homeostasis and prevent tumorigenesis. However, how stem cell proliferation is properly controlled and in particular how the cell cycle is regulated in stem cells is not fully understood. This study identified Yun as an ISC proliferation regulator from a large-scale RNAi screen. Loss of yun function in progenitors restricts them from proliferating, such that they remain in a quiescent state under normal conditions and during tissue regeneration following acute tissue damage. Yun was shown to act as a scaffold for the PHB complex, and the Yun/PHB complex is regulated by EGFR signaling and functions through E2F1 to sustain proliferation of normal stem cells for tissue homeostasis/regeneration and transformed stem cells in tumorigenesis (Zho, 2022).

    In addition to EGFR signaling, the levels of the Yun/PHB complex are also elevated upon activation of JAK/STAT signaling or in the absence of Notch, indicating that the Yun/PHB complex may also be regulated by JAK/STAT and Notch signaling directly or indirectly. Previous studies and the current data show that EGFR signaling acts downstream of JAK/STAT, Notch, and Wnt signaling in ISC proliferation and that ectopic expression of yun/Phb could rescue proliferation defects in the absence of EGFR signaling. Therefore, it is proposed that EGFR signaling is the major upstream of signal of the Yun/PHB complex, although the possibilities cannot be fully excluded that the complex may also be regulated by the other signaling pathways directly or indirectly. The EGFR/MAPK pathway and E2F1 are differentially required for stem cell proliferation (mitosis) and differentiation (endoreplication). How E2F1 is differentially regulated during these two processes is not clear. The regulation of E2F1 levels by EGFR/MAPK signaling has been proposed to be due to increased translation or/and increased protein stability, possibly involving some unknown cytoplasmic factors. The identification of the Yun/PHB complex may account for the differential regulation of E2F1 by EGFR/MAPK signaling. The Yun/PHB complex is expressed in progenitors and mediates EGFR/MAPK signaling for ISC proliferation but not progeny differentiation, indicating that the Yun/PHB complex is more specifically required for ISC proliferation. Interestingly, a previous study identified another target of EGFR signaling, the transcription factor Sox100B/dSox9B, which has a critical role in progeny differentiation, indicating that the control of the ISC proliferation and progeny differentiation by EGFR/MAKP signaling is likely differentially mediated by different effectors (Zho, 2022).

    The levels of E2F1 protein, along with the expression of PCNA-GFP, were significantly diminished in yun/Phb-defective progenitors or imaginal wing discs, suggesting that Yun affects E2F1 levels. Biochemical analysis shows that the Yun/PHB complex associates with E2F1 in vivo, indicating that the Yun/PHB complex interacts and stabilizes E2F1 protein to regulate ISC proliferation. Furthermore, ectopic expression of single components of the Yun/PHB complex increased E2F1 protein levels, which were further increased when two or three components of the Yun/PHB complex were coexpressed, supporting the notion that the Yun/PHB complex regulates E2F1 protein levels. Moreover, ectopic expression of E2F1/Dp significantly restored ISC proliferation in yun/Phb-defective intestines. Consistently, knockdown of the negative regulator of E2F1, Rb, also restored ISC proliferation in these yun/Phb-defective intestines, albeit at a weaker level than that of E2F1/Dp overexpression. Together, these data demonstrate that E2F1 acts downstream of the Yun/PHB complex for ISC proliferation. Interestingly, overexpressing PCNA alone is not sufficient to restore ISC proliferation in yun-depleted intestines, indicating that collective activation of multiple downstream targets of the E2F1/Dp complex are required to restore ISC proliferation. It has been previously proposed that PHB1 binds to Rb and functions as a negative regulator of E2F1-mediated transcription. These studies contrast with previous work suggesting that the PHB complex is required for cell proliferation. This in vivo study uncovered how E2F1 is differential regulated by EGFR/MAPK signaling and acts downstream of the Yun/PHB complex in ISC proliferation to maintain tissue homeostasis under normal and stress conditions and during tumorigenesis in Drosophila, which is in striking contrast to the proposed antiproliferation role of PHB1 (Zho, 2022).

    Unlike Yun, Phb1 and Phb2 are conserved and have been reported to form a complex that localizes to the nucleus, plasma membrane, and mitochondria in mammalian cells. In mitochondria, the PHB complex functions as chaperones in mitochondria of some cell types. Although in flies, the Yun/PHB complex is partially localized in mitochondria in progenitors, the results indicate that the ISC proliferation defects observed in yun/Phb-defective progenitors are unlikely due to their roles in mitochondria. Human Phb1 and Phb2 could significantly restore ISC proliferation defects in Phb1- and Phb2-depleted intestines, respectively, and were required for the proliferation of different human cancer cell lines, indicating that the function of the PHB complex in proliferation is conserved. Finally, the observations that 1) Yun acts as a scaffold of PHBs for their proper function; 2) the Yun/PHB complex acts downstream of EGFR/MAPK signaling; and 3) PHBs and EGFR/MAPK signaling are evolutionarily conserved, suggest that a functional counterpart of Yun exists in mammals, which is different in primary sequence but possibly similar in structure (Zho, 2022).

    Sequential Ras/MAPK and PI3K/AKT/mTOR pathways recruitment drives basal extrusion in the prostate-like gland of Drosophila

    One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. However, due to lack of adequate in vivo model, implication of emblematic signalling pathways such as Ras/Mitogen-Activated Protein Kinase (MAPK) and phosphoinositide 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathways, is scarcely described in this phenomenon. This paper reports a unique model of basal extrusion in the Drosophila accessory gland. There, it was demonstrated that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, this study shows that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer (Rambur, 2020).

    Worldwide, a large majority of cancers originates from epithelial tissues such as lung, breast and prostate1. Despite reinforced prevention, most of the tumours are detected at late stages, and patient care is centred on invasive adenocarcinomas, resistant forms of these carcinomas and metastatic carcinomas. As late stages of cancer progression have been under intense scrutiny in the last decades, the molecular mechanisms associated to such progression are largely described, showing for example the major role of receptor tyrosine kinase (RTK)-dependent signalling pathways in these mechanisms. Typically, for prostate adenocarcinoma, the second most common cancer in men, both PI3K/AKT/mTOR pathway and Ras/MAPK pathways are associated with tumour progression. In the prostate adenocarcinoma, Ras/MAPK and PI3K/AKT/mTOR pathways display activating genetic alterations in more than 40% of primary tumours and in virtually all metastatic prostate tumours, and phosphoproteomic studies confirmed a strong correlation in the activation of these two pathways. Furthermore, pre-clinical mouse models reproducing alteration of either one or the other pathway in the prostate epithelium display tumourigenesis that mimics histopathological features of the human adenocarcinoma. Moreover, advanced tumour progression is obtained when combining alterations in both pathways. These different data emphasise that in one hand, Ras/MAPK or PI3K/AKT/mTOR pathways can initiate prostate tumour development, and in the other hand that these pathways are implicated in late phases of tumour progression. However, they explain neither their respective or combined role in actual adenocarcinoma formation nor the molecular mechanisms that could couple these two pathways to promote this phenomenon in vivo (Rambur, 2020).

    Adenocarcinoma formation occurs when pre-invasive epithelial cells acquire the ability to leave their epithelial compartment. This implicates that these cells are able to extrude from the normal epithelium and to cross the basement membrane which is the limit of the epithelial compartment. These phenomena can be described as basal extrusion and are resulting in early invasion, as opposed to late invasion associated to the metastatic process. Due to the difficulty to precisely visualise basal extrusion in animals, mechanistic associated to this phenomenon has been essentially described in cellular models or in developing tissues such as Drosophila imaginal disc of zebrafish embryo, even though the role of P120 catenin in basal extrusion has been shown in a mouse model of pancreatic neoplasia. The role of Ras/MAPK pathway in basal extrusion has only been described through the use of RasV12 as an oncogenic hit, and role of PI3K/AKT/mTOR pathway has never been assessed (Rambur, 2020).

    To determine the role of Ras/MAPK and PI3K/AKT/mTOR pathways in basal extrusion and understand the underlying mechanisms that may coordinate their hyperactivation in prostate cancer, this study has developed a new model of in vivo early invasive adenocarcinoma in the Drosophila prostate-like accessory gland. Drosophila is a powerful genetic model where more than 70% of genes implicated in human diseases display orthologs and where Ras/MAPK and PI3K/AKT/mTOR signalling pathways are well conserved. Drosophila has already proven its pertinence as cancer model for brain, lung, and colon. The Drosophila accessory gland is a functional equivalent for the prostate, playing a role in fertility by secreting seminal fluid. Secretions come from a monolayer of epithelial cells that are well differentiated and quiescent at the adult age, and there is no evidence of stem cells in this tissue. Considering that a majority of prostate adenocarcinoma is thought to originate from luminal cells, epithelial cells from the accessory gland represent a valuable model to study the mechanisms of epithelial prostate tumourigenesis (Rambur, 2020).

    This study describes this unique model of basal extrusion and tumour formation in the accessory gland that recapitulates most aspects of cancer development. Both Ras/MAPK and PI3K/Akt/TOR pathways are overactivated in the produced tumours, and these pathways cooperate to induce basal extrusion and subsequent tumour formation. Furthermore, the mechanism is described that allows the coactivation of these pathways, which relies on the sequential recruitment of a double autocrine feedback loop dependent on Epidermal (EGF/Spitz) and Insulin-like (IGF/Ilp6) Growth Factors and their respective receptors. Finally, using publicly available data of prostate cancer samples and migration assay in human pre-tumoural prostate epithelial cell line, the possible role of these findings in the actual human pathology is assessed (Rambur, 2020).

    To faithfully reproduce what is thought to happen in the earliest stages of tumour formation in patients, a single genetic alteration was produced in few clones of randomly selected and mostly differentiated cells. Furthermore, accessory gland epithelium, shown to be adjacent to a basement membrane, is surrounded by a stromal-like sheet of muscle fibres, and oncogene-induced epithelial cells are able to cross both layers to form external tumours. This recapitulates the phenomenon of basal epithelial cell extrusion, which is thought to be central to cell invasion. Basal extrusion has been described in cell culture, in Drosophila imaginal discs, in zebrafish embryos and in mouse. However, implication of Ras/MAPK and PI3K/AKT/mTOR pathways has never been assessed in this phenomenon, despite the fact that these pathways are among the most deregulated in cancers, and especially in epithelial cancers such as prostate adenocarcinoma. This study shows in a new model of accessory gland tumourigenesis that both pathways are implicated in basal extrusion, indicating that this step demands a particular state of activation for the cell that undergoes this basal extrusion. Furthermore, this finding correlates with the fact that the two considered pathways are already frequently co-deregulated in primary tumours. From these experiments, where oncogene expression is restricted to few cells and intra-tumoural inhibition of the pathways decreases invasion, it is infered that the mechanisms of basal cell extrusion are cell autonomous, as previously shown in cell lines. Indeed, this study shows that this cell-autonomous mechanism relies on the production of two growth factors, and subsequent activation of two autocrine loops. Role of autocrine loops has been hypothetized in late tumourigenesis, as higher levels of growth factors have been found in tumoural tissues, and has been studied in cell models where inhibition of these loops decreases tumourigenic features such as migration or proliferation capacity as their activation have been linked to transformation of various epithelial cells. However, a role of autocrine loops has never been demonstrated for basal extrusion in vivo. If these loops seem implicated in tumour late progression, so could they be more important for early human tumour development. In fact, many strategies have been attempted to treat cancer patients especially by blocking EGF/EGFR autocrine loop. However, for advanced prostate cancer, these strategies have shown poor results, as well for monotherapies as for combined treatments with classical anti-prostate cancer agents. It could be logical if autocrine loops are less implicated in late stages of cancer but more in the capacity for tumour cells to leave the epithelial compartment. In later stages, higher rates of activating mutations in the Ras/MAPK and PI3K/AKT/mTOR pathways could suppress the need for RTK-driven activation. In contrast, in early tumourigenesis, as fewer genetic alterations are present, activation of signalling pathways must rely on different mechanisms. As is shown in the accessory gland, this recruitment could be efficiently done in tumour cells by autocrine production of growth factors, autocrine activation of their RTK and subsequent activation of the pathways necessary for the tumour development. In a human cohort of prostate cancer samples, it was found that EGF is more expressed in primary tumours than either in normal tissue or in metastases. This could correlate with an early requirement for such growth factor in the formation of adenocarcinoma. Contrary to the observations in Drosophila, no early overexpression of IGFs can be detected in human samples. However, in human, EGFR is able to recruit both Ras/MAPK and PI3K/AKT/mTOR pathway, and EGF overexpression could drive their activation and act in the same way as EGF/Spitz and IGF/Ilp6 in Drosophila (Rambur, 2020).

    To study early phases of tumourigenesis remains difficult in vivo, especially for epithelial cells that can develop into benign tumours still in the epithelial compartment such as benign prostatic intraepithelial neoplasia, or into adenocarcinoma that are characterised by an expansion out of the epithelial compartment. The model developed in the Drosophila accessory gland represents a unique in vivo model to explore basal extrusion and early invasion. Two major pathways of cancer progression are implicated in this basal extrusion, and these two pathways are co-recruited by autocrine loops. Further investigation will be necessary to test whether other pathways implicated in late tumourigenesis are important in this phenomenon. Furthermore, it will also be important to determine which genes are activated or inhibited by these pathways and which mechanisms are recruited to promote the actual extrusion (Rambur, 2020).

    Drosophila RASopathy models identify disease subtype differences and biomarkers of drug efficacy

    RASopathies represent a family of mostly autosomal dominant diseases that are caused by missense variants in the rat sarcoma viral oncogene/mitogen activated protein kinase (RAS/MAPK) pathway including KRAS, NRAS, BRAF, RAF1, and SHP2. These variants are associated with overlapping but distinct phenotypes that affect the heart, craniofacial, skeletal, lymphatic, and nervous systems. This study reports an analysis of 13 Drosophila transgenic lines, each expressing a different human RASopathy isoform. Similar to their human counterparts, each Drosophila line displayed common aspects but also important differences including distinct signaling pathways such as the Hippo and SAPK/JNK signaling networks. Multiple classes of clinically relevant drugs-including statins and histone deacetylase inhibitors-were identified that improved viability across most RASopathy lines; in contrast, several canonical RAS pathway inhibitors proved less broadly effective. Overall, this study compares and contrasts a large number of RASopathy-associated variants including their therapeutic responses (Das, 2021).

    Spratford, C. M., Goins, L. M., Chi, F., Girard, J. R., Macias, S. N., Ho, V. W. and Banerjee, U. (2021). Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants. Development 148(24). PubMed ID: 34918741

    Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants

    Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated owing to lack of appropriate genetic tools. This study reports a Split GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, through their dynamic control of the Notch ligand Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems (Spratford, 2021).

    Progenitor and differentiated cell types have been well described in Drosophila hematopoiesis. Genetic evidence suggested that certain cells have an intermediate characteristic in that they express some progenitor as well as mature cell markers. Although these cells could be identified during Drosophila hematopoiesis owing to their overlapping expression patterns, the absence of tools to directly detect such populations has thus far prevented a detailed analysis of these transitional cells. These cells have been designated IPs and they bridge medullary zone (MZ) progenitors with the cortical zone (CZ) hemocytes. This study used a Split GAL4 strategy to generate CHIZ-GAL4 that allows identification and genetical manipulation of IPs. The IPs of the intermediate zone (IZ) represent a unique cell type that have some characteristics that are distinct from and others that are similar to the cells of the MZ and CZ. For example, IPs express dome, but not E-cad, both of which are MZ markers. Similarly, IPs express Hemolectin (Hml), but not the maturity markers P1 (plasmatocytes) and Hnt (crystal cells). Interestingly, the IP cells share the property of multipotency with cells of the MZ in that both can contribute to all three populations of mature hemocytes. Importantly, it is believed IPs are a unique cell type, as their numbers can be expanded or reduced upon genetic manipulation as shown, for example, with modulation of the Ras pathway. In addition, bulk and single cell RNA-seq data obtained recently in the laboratory identifies several genes that are highly enriched within IPs when compared with their expression in all other cell types in the LG. In future studies, these will serve well as specific IZ markers and provide further functional relevance for this population (Spratford, 2021).

    The MZ cells are fairly quiescent; they are largely held in G2, and will undergo mitosis in a limited number of cells. In contrast, IP cells are found in G1, S and G2 but with a very limited extent of mitosis. It is proposed that before entering the IP state, a dome+ progenitor is released from G2 and it undergoes mitosis. Subsequently, Hml is initiated and continues to be expressed as IPs progress through G1, S and G2. At this point dome expression ceases, thus ending the CHIZ-state. The dome-negative post-CHIZ cell likely undergoes a round of mitosis before it progresses to a differentiated state. The IPs are multipotent and contribute to all of the three mature hemocyte populations. It should be noted that the data presented in this study do not preclude the possibility that a few of the hemocytes might form by a parallel mechanism that does not involve the IPs (Spratford, 2021).

    As in many developmental systems, entry into a proliferative state and fate determination are intimately intertwined and this applies as well to the transition from the IZ to the CZ. It is presumed that a mitotic event must closely follow exit from the IP state and is linked to differentiation into a hemocyte. It is also known that the Ras/Raf pathway is required for exit out of the IP state. In other systems, Ras/Raf activity has largely been associated with proliferation, but in Drosophila, this pathway often governs cell fate determination, as seen, for example, during the development of the eye imaginal disc. Thus, it remains uncertain at the present moment whether Ras/Raf initiates the mitotic process and this allows differentiation signals to be sensed to turn on markers, or whether another mechanism controls the entry into mitosis and Ras is responsible for turning off a marker such as dome. In a manner similar to that seen in other well-defined developmental situations in Drosophila, the Ras/Raf and Notch pathways play dueling roles in the post-CHIZ stage of defining cell fate. The IPs express Ser in a dynamic pattern and induce neighbors to take on a crystal cell fate. The expression of Ser is downregulated after the mid-third instar, and its restricted spatial and temporal pattern of expression limits crystal cell number. Crystal cells do not have active Ras signaling as established by their expression of the Yan protein. The Ras/Raf signal promotes plasmatocyte fate, whereas crystal cells are dependent on Notch signaling. Upstream events that activate Ras in the IPs are currently unknown and will be of great interest for future investigation. It is possible that a canonical ligand-dependent RTK may be involved; however, other autonomous molecular mechanisms such as changes in metabolism could feed into Ras (Spratford, 2021).

    IPs may provide an opportunity to synchronize the assignment of cell fate during normal development, and maintain plasmatocytes and crystal cells in a stereotypical ratio. It is also likely that IPs have unique signaling functions as inferred from their regulation of Ser expression to induce direct neighbors to take on a crystal-cell fate. It is interesting to note that this transitional population acts autonomously as multipotent progenitors while they also non-autonomously induce one of the specific blood cell fates. Investigation into the expression of receptors and ligands in IPs will expand current understanding of the role these cells play in regulating the balance between progenitors and the various determined blood cell types during homeostasis. If all progenitors in the MZ were to directly differentiate into mature hemocytes without going through the buffer zone provided by the IPs, then a relatively steady pool of progenitors will be difficult to preserve, and the spatio-temporal order of hemocyte specification will not be maintained. Under stress conditions or immune challenge this buffer could be altered in favor of faster production of hemocytes at the cost of progenitor number (Spratford, 2021).

    The experimental strategy used to develop CHIZ-GAL4 has been successfully adapted for identifying cell types based on the co-expression of other genes in Drosophila, particularly in the nervous system. There is nothing about this strategy that is Drosophila-specific and one hopes that its most useful application might be to uncover cryptic cell types in the context of the significantly more complex transitions described in mammalian hematopoietic development (Spratford, 2021).

    proteins associated with the ras pathway


    References

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    Zygotically transcribed genes

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