rolled/MAPK: | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - rolled

Synonyms - MAP kinase

Cytological map position - h38--h41

Function - serine/threonine protein kinase

Keywords - Ras pathway, Terminal group, Eye, FGF signaling

Symbol - rl

FlyBase ID:FBgn0003256

Genetic map position - 2-55.1

Classification - Map kinase - ERK

Cellular location - cytoplasmic and nuclear

NCBI link: Entrez Gene
rl orthologs: Biolitmine

Recent literature
Kushnir, T., Bar-Cohen, S., Mooshayef, N., Lange, R., Bar-Sinai, A., Rozen, H., Salzberg, A., Engelberg, D. and Paroush, Z. (2019). An activating mutation in ERK causes hyperplastic tumors in a scribble mutant tissue in Drosophila. Genetics. PubMed ID: 31740452
Receptor tyrosine kinase signaling plays prominent roles in tumorigenesis, and activating oncogenic point mutations in the core pathway components Ras, Raf or MEK are prevalent in many types of cancer. Intriguingly, however, analogous oncogenic mutations in the downstream effector kinase ERK have not been described or validated in vivo. To determine if a point mutation could render ERK intrinsically active and oncogenic, this study has assayed in Drosophila the effects of a mutation that confers constitutive activity upon a yeast ERK ortholog, and which has been also identified in a few human tumors. These analyses indicate that a fly ERK ortholog harboring this mutation alone (RolledR80S), and more so in conjunction with the known sevenmaker mutation (RolledR80S+D334N), suppresses multiple phenotypes caused by loss of Ras-Raf-MEK pathway activity, consistent with an intrinsic activity that is independent of upstream signaling. Moreover, expression of RolledR80S and RolledR80S+D334N induces tissue overgrowth in an established Drosophila cancer model. These findings thus demonstrate that activating mutations can bestow ERK with pro-proliferative, tumorigenic capabilities and suggest that Drosophila represents an effective experimental system for determining the oncogenicity of ERK mutants and their response to therapy.
Ahlers, L. R. H., Trammell, C. E., Carrell, G. F., Mackinnon, S., Torrevillas, B. K., Chow, C. Y., Luckhart, S. and Goodman, A. G. (2019). Insulin potentiates JAK/STAT signaling to broadly inhibit Flavivirus replication in insect vectors. Cell Rep 29(7): 1946-1960.e1945. PubMed ID: 31722209
The World Health Organization estimates that more than half of the world's population is at risk for vector-borne diseases, including arboviruses. Because many arboviruses are mosquito borne, investigation of the insect immune response will help identify targets to reduce the spread of arboviruses. This study used a genetic screening approach to identify an insulin-like receptor as a component of the immune response to arboviral infection. It was determined that vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, we show that insulin signaling activates the JAK/STAT, but not RNAi, pathway via ERK to control infection in Drosophila cells and Culex mosquitoes through an integrated immune response. Finally, insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to human pathogens.
Patel, A. L., Yeung, E., McGuire, S. E., Wu, A. Y., Toettcher, J. E., Burdine, R. D. and Shvartsman, S. Y. (2019). Optimizing photoswitchable MEK. Proc Natl Acad Sci U S A 116(51): 25756-25763. PubMed ID: 31796593
Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. This study presents evidence that psMEK indeed has suboptimal functionality in vivo, and a strategy is proposed to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, it was demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. It was establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, it esd udrf to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.
Yu, J., Zheng, Q., Li, Z., Wu, Y., Fu, Y., Wu, X., Lin, D., Shen, C., Zheng, B. and Sun, F. (2021). CG6015 controls spermatogonia transit-amplifying divisions by epidermal growth factor receptor signaling in Drosophila testes. Cell Death Dis 12(5): 491. PubMed ID: 33990549
Spermatogonia transit-amplifying (TA) divisions are crucial for the differentiation of germline stem cell daughters. However, the underlying mechanism is largely unknown. The present study demonstrated that CG6015 was essential for spermatogonia TA-divisions and elongated spermatozoon development in Drosophila melanogaster. Spermatogonia deficient in CG6015 inhibited germline differentiation leading to the accumulation of undifferentiated cell populations. Transcriptome profiling using RNA sequencing indicated that CG6015 was involved in spermatogenesis, spermatid differentiation, and metabolic processes. Gene Set Enrichment Analysis (GSEA) revealed the relationship between CG6015 and the epidermal growth factor receptor (EGFR) signaling pathway. Unexpectedly, it was discovered that phosphorylated extracellular regulated kinase (dpERK) signals were activated in germline stem cell (GSC)-like cells after reduction of CG6015 in spermatogonia. Moreover, Downstream of raf1 (Dsor1), a key downstream target of EGFR, mimicked the phenotype of CG6015, and germline dpERK signals were activated in spermatogonia of Dsor1 RNAi testes. Together, these findings revealed a potential regulatory mechanism of CG6015 via EGFR signaling during spermatogonia TA-divisions in Drosophila testes.
Zheng, Q., Chen, X., Qiao, C., Wang, M., Chen, W., Luan, X., Yan, Y., Shen, C., Fang, J., Hu, X., Zheng, B., Wu, Y. and Yu, J. (2021). Somatic CG6015 mediates cyst stem cell maintenance and germline stem cell differentiation via EGFR signaling in Drosophila testes. Cell Death Discov 7(1): 68. PubMed ID: 33824283
Stem cell niche is regulated by intrinsic and extrinsic factors. In the Drosophila testis, cyst stem cells (CySCs) support the differentiation of germline stem cells (GSCs). However, the underlying mechanisms remain unclear. This study found that somatic CG6015 is required for CySC maintenance and GSC differentiation in a Drosophila model. Knockdown of CG6015 in CySCs caused aberrant activation of dpERK in undifferentiated germ cells in the Drosophila testis, and disruption of key downstream targets of EGFR signaling (Dsor1 and rl) in CySCs results in a phenotype resembling that of CG6015 knockdown. CG6015, Dsor1, and rl are essential for the survival of Drosophila cell line Schneider 2 (S2) cells. The data showed that somatic CG6015 regulates CySC maintenance and GSC differentiation via EGFR signaling, and inhibits aberrant activation of germline dpERK signals. These findings indicate regulatory mechanisms of stem cell niche homeostasis in the Drosophila testis.
Yuen, A. C., Prasad, A. R., Fernandes, V. M. and Amoyel, M. (2022). A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila. Biol Open 11(5). PubMed ID: 35608229
Extracellular signal-regulated kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. This study adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, this study made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active epidermal growth factor receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, the direction of migration of retinal basal glia correlated with their ERK activity levels, suggesting an explanation for the heterogeneity in ERK activity observed in fixed tissue. These results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.
Leahy, S. N., Song, C., Vita, D. J. and Broadie, K. (2023). FMRP activity and control of Csw/SHP2 translation regulate MAPK-dependent synaptic transmission. PLoS Biol 21(1): e3001969. PubMed ID: 36701299
Noonan syndrome (NS) and NS with multiple lentigines (NSML) cognitive dysfunction are linked to SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) gain-of-function (GoF) and loss-of-function (LoF), respectively. In Drosophila disease models, this study found both SHP2 mutations from human patients and corkscrew (csw) homolog LoF/GoF elevate glutamatergic transmission. Cell-targeted RNAi and neurotransmitter release analyses reveal a presynaptic requirement. Consistently, all mutants exhibit reduced synaptic depression during high-frequency stimulation. Both LoF and GoF mutants also show impaired synaptic plasticity, including reduced facilitation, augmentation, and post-tetanic potentiation. NS/NSML diseases are characterized by elevated MAPK/ERK signaling, and drugs suppressing this signaling restore normal neurotransmission in mutants. Fragile X syndrome (FXS) is likewise characterized by elevated MAPK/ERK signaling. Fragile X Mental Retardation Protein (FMRP) binds csw mRNA and neuronal Csw protein is elevated in Drosophila fragile X mental retardation 1 (dfmr1) nulls. Moreover, phosphorylated ERK (pERK) is increased in dfmr1 and csw null presynaptic boutons. Presynaptic pERK activation was found in response to stimulation is reduced in dfmr1 and csw nulls. Trans-heterozygous csw/+; dfmr1/+ recapitulate elevated presynaptic pERK activation and function, showing FMRP and Csw/SHP2 act within the same signaling pathway. Thus, a FMRP and SHP2 MAPK/ERK regulative mechanism controls basal and activity-dependent neurotransmission strength.
Taylor, C. A. t., Cormier, K. W., Martin-Vega, A., Earnest, S., Stippec, S., Wichaidit, C. and Cobb, M. H. (2023). ERK2 Mutations Affect Interactions, Localization, and Dimerization. Biochemistry. PubMed ID: 37021821
The most frequent ERK2 (MAPK1; see Drosophila Rolled) mutation in cancers, E322K, lies in the common docking (CD) site, which binds short motifs made up of basic and hydrophobic residues present in the activators MEK1 (MAP2K1) and MEK2 (MAP2K2), in dual specificity phosphatases (DUSPs) that inactivate the kinases, and in many of their substrates. Also, part of the CD site, but mutated less often in cancers, is the preceding aspartate (D321N). These mutants were categorized as gain of function in a sensitized melanoma system. In Drosophila developmental assays, this study found that the aspartate but not the glutamate mutant caused gain-of-function phenotypes. This study catalogued additional properties of these mutants to accrue greater insight into their functions. A modest increase in nuclear retention of E322K was noted. Binding of ERK2 E322K and D321N to a small group of substrates and regulatory proteins was similar, in spite of differences in CD site integrity. Interactions with a second docking site, the F site, which should be more accessible in E322K, were modestly reduced rather than increased. The crystal structure of ERK2 E322K also indicated a disturbed dimer interface, and reduced dimerization was detected by a two-hybrid test; yet, it was detected in dimers in EGF-treated cells, although to a lesser extent than D321N or wt ERK2. These findings indicate a range of small differences in behaviors that may contribute to increased function of E322K in certain cancers.
Ho, E. K., Oatman, H. R., McFann, S. E., Yang, L., Johnson, H. E., Shvartsman, S. Y. and Toettcher, J. E. (2023). Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo. bioRxiv. PubMed ID: 36945584
Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. This study used optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. High or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that an all-or-none stimulus can be 'blurred' through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, this study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.
Sung, H., Vaziri, A., Wilinski, D., Woerner, R. K. R., Freddolino, P. L. and Dus, M. (2023). Nutrigenomic regulation of sensory plasticity. Elife 12. PubMed ID: 36951889
Diet profoundly influences brain physiology, but how metabolic information is transmuted into neural activity and behavior changes remains elusive. This study shows that the metabolic enzyme O-GlcNAc Transferase (OGT) moonlights on the chromatin of the D. melanogaster gustatory neurons to instruct changes in chromatin accessibility and transcription that underlie sensory adaptations to a high-sugar diet. OGT works synergistically with the Mitogen Activated Kinase/Extracellular signal Regulated Kinase (MAPK/ERK) rolledand its effector stripe (also known as EGR2 or Krox20) to integrate activity information. OGT also cooperates with the epigenetic silencer Polycomb Repressive Complex 2.1 (PRC2.1) to decrease chromatin accessibility and repress transcription in the high-sugar diet. This integration of nutritional and activity information changes the taste neurons' responses to sugar and the flies' ability to sense sweetness. These findings reveal how nutrigenomic signaling generates neural activity and behavior in response to dietary changes in the sensory neurons.
Ho, E. K., Oatman, H. R., McFann, S. E., Yang, L., Johnson, H. E., Shvartsman, S. Y. and Toettcher, J. E. (2023). Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo. Development. PubMed ID: 37602510
Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. This study use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. High or low doses of ERK signaling produce transient or sustained byn expression, respectively. While tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that "blurring" of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, this study provides a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.
Yue, W., Deng, X., Wang, Z., Jiang, M., Hu, R., Duan, Y., Wang, Q., Cui, J. and Fang, Y. (2023). Inhibition of the MEK/ERK pathway suppresses immune overactivation and mitigates TDP-43 toxicity in a Drosophila model of ALS. Immun Ageing 20(1): 27. PubMed ID: 37340309 TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using Drosophila as a model, this study uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). A robust immune overactivation was pbserved in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, this study showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, these findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases (Yue, 2023).
Wilcockson, S. G., Guglielmi, L., Araguas Rodriguez, P., Amoyel, M., Hill, C. S. (2023). An improved Erk biosensor detects oscillatory Erk dynamics driven by mitotic erasure during early development. Dev Cell, 58(23):2802-2818.e2805 PubMed ID: 37714159
Extracellular signal-regulated kinase (Erk) signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics in vivo, as a gradient of activated diphosphorylated Erk (P-Erk) is induced by fibroblast growth factor (Fgf) signaling at the blastula margin. This study describes an improved Erk-specific biosensor, which we term modified Erk kinase translocation reporter (modErk-KTR). The utility of this biosensor was demonstrated in vitro and in developing zebrafish and Drosophila embryos. Moreover, it was shown that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Erk activity is rapidly extinguished just prior to mitosis, which is referred to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. These modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics in vivo.

rolled/MAP kinase is essential to the proper functioning of the Ras signaling pathway. Mammalian homologs are known as ERKs (extracellular signal-regulated kinases) because of their roles in transducing signals from outside the cell into the nucleus. Targets of Rolled in Drosophila include Pointed, Anterior open (commonly referred to as Yan), Seven in absentia (Sina) and Jun related antigen (Jun or DJun). For example, although induction of Sina is not regulated by Rolled, its biological activity is regulated by Ras pathway phosphorylation (Dickson, 1992). The same holds true for Anterior open. Anterior open activity is targeted by the Ras pathway (Gabay, 1996). Likewise, the Ras pathway activates Pointed protein (Brunner, 1994b) and Jun related antigen, commonly known as Jun (Peverali,1996).

The development of the Drosophila eye is controlled in part, by the Ras pathway. This provides a good illustration of rolled/Mapk functions. The Drosophila compound eye is made up of a cluster of single simplified eye elements, collectively termed ommatidia. Each ommatidium contains eight photoreceptors (R1-R8), differentiated during larval development in a process of discrete steps from the larval eye imaginal disc: in order of determination, they are R8, followed by the pairs R2/R5, R3/R4, and R1/R6, and finally R7. Induction of R7 involves signals from the R8 photoreceptor. The R8 photoreceptor presents on its surface a ligand, Bride of Sevenless, that binds and activates Sevenless receptor tyrosine kinase in the R7 precursor. Autophosphorylated Sevenless initiates a Ras1-mediated cascade, which eventually activates transcription factors in the nucleus via Raf1 and MAP kinases, resulting in R7 development (Yamamoto, 1994 and references).

The presence of functional R7 photoreceptor cells can be detected by a simple behavioral test. Given a choice between an ultraviolet (UV) and a visible light source, wild-type flies will move towards the UV light. Flies lacking R7 cells, however, will move towards the visible light. This behavior has been used to identify mutations in genes that prevent the development of R7 photoreceptor cells and has led to the identification of sevenless and bride of sevenless genes. This behavioral screen can be used to identify dominant mutations that result in the activation of the sevenless signal transduction pathway, even in the absence of its inducing signal, the Boss protein (Brunner, 1994a and references).

Sevenmaker, a gain of function rolled mutation, specifies R7 photoreceptor cells independently of boss and sev functions. In boss mutant flies, the R7 cell is missing, whereas in Sev gain of function mutants many ommatidia contain multiple R7-like cells (Brunner, 1994a). Rolled acts downstream of Ras1 and Raf in specifying the R7 fate. In addition, rolled plays a key role in the Torso pathway. Gain of function Sev results in the ectopic acivation of the Torso signaling pathway in a manner similar to its effect on the sevenless pathway in the developing eye. Loss-of-function mutations in rolled can suppress gain-of-function torso mutations. Thus Rolled acts in the terminal pathway responsible for specification of terminal structures immediately after fertilization. Likewise rolled mutations affect signaling of the Drosophila Epidermal growth factor receptor. Gain-of-function Sevenmaker mutations produce extra wing veins close to the wing margin. In addition, rolled mutations modify EGF-R signaling in the establishment of the dorsoventral polarity of the egg shell and the embryo (Brunner, 1994a).

Rolled activity is targeted during heat shock induced stress in cultured Drosophila cells. Heat shock activates a MAP kinase phosphatase, resulting in a reduction of Rolled activity over the course of a 15 minute heat shock regime (raising the temperature from 25 to 37 degrees) (Cornelius, 1995).

Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still poorly understood. The survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein Hid via the Egf receptor/RAS/MAPK pathway. The TGF alpha-like ligand Spitz is activated in the neurons, and glial cells compete for limited amounts of secreted Spitz to survive. In midline glia that fail to activate the Egfr pathway, Hid induces apoptosis by blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established (Bergmann, 2002).
The reduction in midline glia (MG) cell number due to apoptosis, as well as the requirement of the RAS/MAPK pathway for MG survival, has been documented using various MG-specific enhancer trap lines and reporter fusion constructs. The MG are visualized using a reporter fusion construct for the slit gene (sli-lacZ) in which a 1 kb fragment of the slit promoter confers expression specifically to the MG. Using a ß-gal antibody to monitor the developmental profile of the MG, about ten cells per segment expressing sli-lacZ are detectable at midembryogenesis (stage 13). By the end of embryogenesis at stage 17, the number of sli-lacZ-positive cells is reduced to approximately three per segment. Since the sli-lacZ expression is specific for the MG, sli-lacZ-expressing cells are referred to as MG (Bergmann, 2002).

Prominent activation of MAPK has been identified in the MG cells, but its functional role has not been determined. The fate of the MG in mapk-deficient embryos has been analyzed. Compared to wild-type embryos, the initial generation of the MG appears to be normal. However, by stage 17 (the end of embryogenesis), none of the MG in mapk-deficient embryos survive. This finding suggests that MAPK is required for MG survival in wild-type embryos (Bergmann, 2002).

The genetic requirement of mapk for MG survival and of hid for MG apoptosis prompted the assumption that MAPK promotes survival of the MG by inhibition of HID activity. According to this model, the MG would be unprotected from HID-induced apoptosis in mapk-deficient embryos, and die. Consistent with this idea, HID protein is detectable in the MG of late stage wild-type embryos. To test this further, embryos that were mutant for both mapk and hid were examined. In early stage mapk;hid double mutant embryos, the initial generation of the MG appears to be normal. However, in contrast to mapk mutants alone, the MG is rescued in mapk;hid double mutant embryos although the survival function of MAPK is missing in these embryos. Dissection revealed that the MG are located directly at the cuticle of the embryos. Because segmental fusions occur in these embryos, some of the MG cluster in groups of up to 20 cells. In individual segments, five to six MG are visible. This number is larger compared to wild-type (three MG per segment), and is remarkably similar to the number of surviving MG in hid mutant embryos alone, indicating that MAPK promotes MG survival largely through inhibition of HID (Bergmann, 2002).

The mutant analysis revealed that MAPK is required to suppress the activity of HID for MG survival. If hid is mutant in mapk-deficient embryos (i.e., in mapk; hid double mutants), MAPK is no longer needed for the survival of the MG. Thus, MAPK-mediated survival of the MG functions through inhibition of HID (Bergmann, 2002).

In hid mutant embryos there is a 2-fold increase of the MG compared to wild-type. Approximately six MG per segment survive in hid embryos compared to the 2.8 MG per segment in wild-type, indicating that hid is genetically required for MG cell death. MAPK activity is required for MG survival. Does the level of MAPK activity determine the final number of surviving MG cells? Mutational activation of MAPK, using a dominant allele of MAPK termed Sevenmaker or mapkSem, promotes survival of extra MG. About 6.0 MG per segment survive in stage 17 mapkSem embryos, providing additional evidence that MAPK is required for MG survival. Remarkably, the number of surviving MG in mapkSem embryos is very similar to the number of surviving MG in hid mutant embryos. In both cases, approximately six MG survive per segment. Therefore, it was determined whether the six surviving MG in mapkSem embryos correspond to the same MG that survive in hid mutant embryos by double mutant analysis. Stage 17 mapkSem; hid double mutant embryos contain on average 6.6 MG, or slightly more than the single mutants alone. This result strongly suggests that hid expression and MAPK activation occur in largely the same set of MG, that is, in a group of about six MG. If MAPK activation and hid expression would occur in different MG independently of each other, then the mapkSem;hid double mutant would be expected to be the composite of the individual mutants and a total of about ten to twelve MG would survive in the double mutant, similar to what has been observed in H99 mutant embryos. It is inferred from the double mutant analysis that the survival of approximately six MG is regulated by MAPK-dependent inhibition of HID. As long as MAPK is activated, these MG survive (as seen in the activated mapkSem mutant). However, MG in this group that does not maintain activated MAPK are eliminated by HID-induced apoptosis. Thus, the coordinated expression of HID and activation of MAPK regulate the final MG cell number (Bergmann, 2002).

MAPK suppresses hid activity in two ways: via downregulation of its transcription and via phosphorylation of HID protein. However, hid mRNA and protein are readily detectable in the surviving MG of wild-type embryos. Therefore, transcriptional downregulation of hid does not account for MG survival. This prompted a test to see whether inhibitory phosphorylation of HID by MAPK might be critical for MG survival. For this purpose, advantage was taken of an observation that overexpression of HID in the MG using the MG-specific sli-GAL4 driver and UAS-hid transgenes is not sufficient to induce MG apoptosis. Even two copies of the UAS-hid transgenes are not able to ablate the MG. This is contrary to findings in other tissues in which expression of hid induces cell death very well. However, since MAPK is activated in the MG and required for MG survival, it was hypothesized that even overexpressed HID might be inactivated via MAPK phosphorylation (Bergmann, 2002).

To examine this further, UAS-hid transgenes were generated that alter the five phosphoacceptor residues of the MAPK phosphorylation sites to nonphosphorylatable Ala residues (UAS-hidAla5). The UAS-hidAla5 transgenes driven by sli-GAL4 induce apoptosis in the MG very efficiently. One copy of a UAS-hidAla5 transgene is sufficient for the ablation of the MG. Occasionally, some embryos are recovered in which the ablation of the MG is incomplete. However, nerve cord preparations reveal that in these embryos, only a small fraction of the MG survives compared to wild-type. Some segments completely lack MG cells, while others just contain one remaining MG. The MG is required for separation of the commissural axon tracts of the CNS. Consistently, expression of the UAS-hidAla5 transgenes and consequently ablation of the MG causes a fused commissure phenotype. In summary, this analysis demonstrates that MG survival requires suppression of HID activity by MAPK. The MAPK phosphorylation sites in HID are critical for this response, providing an important mechanism for the regulation of MG number (Bergmann, 2002).

Activation of MAPK usually requires activation of RAS, which in turn is activated by receptor tyrosine kinase (RTK) signaling: this demonstrates that MG survival depends on RAS, which is consistent with the model. Within the embryonic CNS, the Drosophila homolog of the Epidermal growth factor receptor (Egfr) is specifically expressed and required for MG differentiation. The requirement of Egfr signaling for MG survival was examined (Bergmann, 2002).

Due to severe developmental defects in egfr mutants, only a few MG start forming at stage 11, and none of them survive. Thus, it is difficult to directly study the requirement of the Egfr for MG survival. To overcome this problem, a dominant-negative mutant of the Egfr (UAS-EgfrDN) was expressed in the MG using the sli-GAL4 driver in otherwise wild-type embryos. In this way, Egfr activity is specifically diminished in the MG after their generation. As expected, the MG form normally in these embryos. However, most of the MG die during subsequent developmental stages and only a few survive to the end of embryogenesis, indicating a direct requirement of the Egfr for MG survival. To determine whether the MG death in this experimental condition is due to failure to inhibit HID, EgfrDN was expressed in the MG of hid mutants. In this genetic background, on average 6.1 MG cells survive, demonstrating that MG survival requires functional Egfr signaling to suppress HID activity (Bergmann, 2002).

The spi gene is required for MG survival and encodes a candidate trophic factor for MG survival. To prove that spi function is required to suppress hid activity, the fate of the MG in spi;hid double mutant embryos was determined. The MG survive in spi embryos if hid is removed as well, and it is concluded that the survival function of spi is mediated through suppression of hid-induced apoptosis (Bergmann, 2002).

The question arises as to which cells process mSPI and provide a source of sSPI for MG survival. Since spi is ubiquitously expressed, it is difficult to determine histochemically where sSPI, the active ligand, is generated. Therefore, a genetic approach was used; whether the loss of MG in spi mutant embryos can be rescued by expression of the membrane bound inactive precursor (UAS-mSPI) either in the MG (using the sim-GAL4 driver) or in neuronal axons (using the elav-GAL4 driver) was examined. It was reasoned that the MG would be rescued in spi mutant embryos only if mSPI is presented in the location where it is normally processed for MG survival in wild-type embryos. Presentation of mSPI by the MG itself does not result in rescue of the MG in spi mutant embryos, ruling out an autocrine mechanism. In contrast, expression of mSPI in neuronal axons appears to be sufficient for MG survival in spi embryos. This argues in favor of a paracrine mechanism. In control experiments, sSPI was examined using these two Gal4 drivers in wild-type embryos. With both GAL4 drivers an increase in the number of MG cells is detected, indicating that they are expressed at the right time and that the MG does not fail to secrete Spi once it has been processed (Bergmann, 2002).

A key regulator of Spi activation is rhomboid, a gene encoding a cell surface, seven-pass transmembrane protein that appears to function as a serine protease directly cleaving mSPI. rhomboid has been implicated in suppression of MG apoptosis. Ectopic expression of Rho in neurons (elav-Gal4/UAS-Rhomboid) promotes an excess of MG, suggesting that neurons have the capacity to process endogenous mSPI. Another essential protein for Spi processing is Star. Star mutants display an MG phenotype similar to spi. STAR regulates intracellular trafficking of mSPI. Expression of Star from the neurons but not from the MG rescues the Star phenotype in the MG. Thus, this analysis clearly demonstrates that the sSPI signal for MG survival is generated and secreted by neurons (Bergmann, 2002).

The surviving MG in late stage embryos are in close contact to commissural axons. In embryos lacking the commissureless (comm) gene, the commissural axons are absent. In comm embryos the MG die prematurely, and some survivors become misplaced laterally along the longitudinal axon tracts. The location of the MG along the longitudinal axons in comm mutant embryos as well as their close contact to commissural axons in wild-type embryos has prompted the suggestion that axon contact is required for MG survival. Axon contact appears to permit the MG to respond to trophic signaling, which is necessary for its survival. Consistent with this notion trophic signaling provided by sSPI/Egfr is present only in MG associated with longitudinal axons in comm;hid double mutants. Thus, it was asked whether axon contact-mediated Egfr signaling in the MG is required to activate MAPK, which in turn suppresses the cell death-inducing ability of HID (Bergmann, 2002).

To address this question, the fate of the MG was examined in comm mutant embryos which are at the same time mutant for hid (comm;hid double mutants) or carry the dominant active mapk allele, mapkSem (mapkSem;comm double mutants). Strikingly, a substantial number of the MG survive even in the absence of axonal contact if hid function is removed or if MAPK is activated. This strongly suggests that axon contact is necessary to suppress HID via MAPK. Only MG in proximity to neurons undergo Egfr signaling. The additional MG that survive along the midline in comm;hid mutants do not express spry, that is, do not receive an Egfr signal, and survive only because hid is absent in this experimental condition (Bergmann, 2002).

It is noted that active MAPK is capable of rescuing a total of six MG based on analysis of mapkSem embryos. Presumably, this MAPK activation in mapkSem embryos is inherited from the differentiation period of the MG. However, only three MG survive by stage 17 in wild-type embryos. It is proposed that of the group of six MG that require MAPK for survival, only the three surviving cells make adequate axon contacts necessary to receive sufficient quantities of the survival factor sSPI. According to this model, the remaining three MG die because they lose the competition for axon contact and do not receive levels of sSPI that are high enough to inactivate HID via phosphorylation by MAPK. If additional sSPI is provided in the midline, additional MG can be rescued. The limited availability of axon-derived sSPI would serve to match the number of MG to the length of commissural axons requiring ensheathment. Thus, the regulation of MG number and survival represents a genetically defined example of the classical trophic theory of cell survival (Bergmann, 2002).

The regulation of MG apoptosis in Drosophila bears striking overall similarity to the regulation of glial cell death in the rat optic nerve. There is an early dependence of the oligodendroglia in the rat optic nerve on growth factors for differentiation followed by a dependence on axon contact for survival. However, it is not clear how the oligodendroglia in the rat optic nerve survive upon axon contact. Since mammalian homologs for many of the components in the apoptotic pathway both upstream and downstream of Drosophila HID are known, it will be interesting to analyze whether similar molecules regulate apoptosis and cell number in the mammalian nervous system. Therefore, molecular genetic studies in Drosophila promise considerable insight for advancing an understanding of the basic control mechanisms involved in the regulation of apoptosis in the context of a developing organism in vivo (Bergmann, 2002).

The rolled locus is found in a heterochromatic region of chromosome 2 that is considered to remain condensed (and for the most part transcriptionally inactive) throughout all or most of the cell cycle. rolled lies in what is considered to be alpha heterochromatin, a chromosome region that makes up the chromocenter of polytene salivary gland chromosomes. The chromocenter is not thought to be polytenized, that is, it is not thought to undergo repeated rounds of DNA replication resulting in multiple copies of active genes. The chromocenter is thought to be made up of DNA and protein in a tightly knit dense structure of which is transcriptionally inactive. Such heterochomatic regions, which make up 30% of the Drosophila genome, have a much lower density of genes as compared to euchromatin.

rolled gene activity is unusual in that it requires the surrounding heterochromatin for gene function. rolled gene activity is severly impaired by bringing rolled close to any euchromatic position; however, these position effects can be reversed by chromosomal rearrangements that bring the rolled gene closer to any block of autosomal or X chromosome heterochromatin (Eberl, 1993). Heterochromatic rolled is extensively polytenized and transcriptionally active in salivary gland chromosomes. rolled undergoes polytenization in salivary gland chromosomes to a degree comparable to that of euchromatic genes, despite its deep heterochromatic location. Sequences on either side of rolled are severly underrepresented in polytene chromosomes and are considered to appear in alpha-heterochromatin. It is suggested that multiple domains of functionally active, complex DNA sequences, composed of single-copy genes and middle-repetitive elements, are present within the proximal heterochromatin of chromosome 2. It may well be that heterochromatin is not the transcriptional desert that it was once thought to be (Berghella, 1996).

Nuclear trapping of ERK shapes the terminal gradient in the Drosophila embryo

Patterning of the terminal regions of the Drosophila embryo relies on the gradient of phosphorylated ERK/MAPK (dpERK), which is controlled by the localized activation of the Torso receptor tyrosine kinase. This model is supported by a large amount of data, but the gradient itself has never been quantified. This study presents the first measurements of the dpERK gradient and establishes a new intracellular layer of its regulation. Based on the quantitative analysis of the spatial pattern of dpERK in mutants with different levels of Torso as well as the dynamics of the wild-type dpERK pattern, it is proposed that the terminal-patterning gradient is controlled by a cascade of diffusion-trapping modules. A ligand-trapping mechanism establishes a sharply localized pattern of the Torso receptor occupancy on the surface of the embryo. Inside the syncytial embryo, nuclei play the role of traps that localize diffusible dpERK. It is argued that the length scale of the terminal-patterning gradient is determined mainly by the intracellular module (Coppey, 2008).

This study identifies the nuclear trapping of dpERK as a mechanism responsible for the intracellular spatial processing of the terminal signal. This conclusion is based on the analysis of the dynamics of the wild-type dpERK gradient. Between nuclear cycles 10 and 14, the dpERK levels are amplified at the termini and attenuated in the subterminal regions of the embryo. The observed dynamics of the dpERK gradient is consistent with a model where dpERK is a diffusible molecule, which is trapped and dephosphorylated by the nuclei. A uniform increase in the nuclear density would increase the trapping of the dpERK molecules at the poles and prevent their diffusion to the middle of the embryo. This is consistent with biochemical and imaging data showing that dpERK rapidly translocates to the nucleus, which can also serve as a compartment of dpERK dephosphorylation. In addition, the model makes a testable prediction about the dynamics of the nucleocytoplasmic (N/C) ratio of phosphorylated MAPK (Coppey, 2008).

The nuclear and cytoplasmic levels of dpERK were quantified in cycle 13 and 14 embryos. Plotting the nuclear and cytoplasmic profiles against each other gives a clear linear relationship, as predicted by a simple formula. Furthermore, the nucleocytoplasmic ratio clearly increases between these two nuclear cycles: the N/C ratio is ~1.4 and ~2 at nuclear cycles 13 and 14, respectively. These measurements show that the nuclear trapping rate is indeed an increasing function of the nuclear density, as predicted by the model (Coppey, 2008).

The observed N/C ratios show that a significant fraction of total dpERK nuclear. As a consequence, defects in the nuclear density should generate clear defects in the gradient. This can be tested in mutants with 'holes' in the nuclear density in blastoderm embryos. For example, in shakleton (shkl) embryos, the migration of nuclei to the poles is delayed and a number of embryos exhibit major disruptions in nuclear density. As predicted by the model, shkl embryos show striking disruptions in the dpERK gradient. The quantified posterior gradient of this particular mutant embryo shows a clear local correlation with the nuclear distribution, emphasizing the role of the nuclei at this stage. In early embryos, the gradient is more extended, presumably reflecting the lack of the nuclei at the poles. Similar defects were found in the giant nuclei (gnu) mutant embryos, which show a different type of defect in nuclear organization. These results support the model in which the syncytial nuclei play an important role in shaping the dpERK gradient (Coppey, 2008).

To summarize, it is proposed that the dpERK gradient is controlled by a cascade of at least two diffusion-trapping modules. In the extracellular compartment, a ligand-trapping mechanism, identified in previous studies, establishes a sharp gradient of Torso receptor occupancy. A similar mechanism regulates the dpERK gradient inside the embryo, where syncytial nuclei act as traps that localize diffusible dpERK. At this time, it cannot be ruled out that the observed sharpening of the dpERK gradient can be modulated also by changes in the spatial distribution of the Torso ligand, but currently there are no data in support of this mode of regulation (Coppey, 2008).

The dynamics of the dpERK gradient is qualitatively different from that of the Bicoid gradient, which remains stable during the last five nuclear divisions. It has been proposed that a stable gradient of Bicoid can be established in the absence of Bicoid degradation, because of the reversible trapping of Bicoid by an exponentially increasing number of nuclei. The differences between the dynamics of the Bicoid and dpERK gradients are attributed to two effects. The first effect is due to the differences in the 'chemistries' of the two systems: the morphogen in the terminal system is degraded (MAPK is dephosphorylated), whereas the anterior morphogen is stable (it is proposed that Bicoid is not degraded on time scale of the gradient formation). The second effect is due to the differences in the initial conditions: by the 10th nuclear division, which is the starting point of the activation of the terminal system, Bicoid gradient is essentially fully established. Thus, a common biophysical framework can describe the Bicoid and dpERK gradients. It remains to be determined whether the nuclear export affects the length scale of the Dorsal gradient, which patterns the dorsoventral axis of the embryo (Coppey, 2008)

ERK phosphorylation regulates sleep and plasticity in Drosophila

Given the relationship between sleep and plasticity, this study examined the role of Extracellular signal-regulated kinase (ERK, Rolled in Drosophila) in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the within the Pigment Dispersing Factor (PDF)-expressing ventral lateral neurons (LNvs) can be recapitulated by expressing an active version of ERK (UAS-ERKSEM) pan-neuronally in the adult fly using GeneSwitch (Gsw) Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE)-luciferase reporter it was shown that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep (Vanderheyden, 2013).

ERK plays a key role in regulating not only cell differentiation and proliferation during development, but is also critical for regulating long-term potentiation and plasticity related events in the fully developed adult. Recent studies have highlighted the important relationship between plasticity induced by waking-experience and sleep need. With that in mind, it was hypothesized that ERK may provide a molecular link between plasticity and sleep. Since, ERK phosphorylation has been previously correlated with sleep time following rhomboid mediated activation of EGFR, this study over-expressed an active version of ERK pan-neuronally in the adult fly and found a significant increase in sleep. ERK activation increased sleep during the day, was rapidly reversible, and was associated with increased activity during waking. In contrast, disrupting ERK signaling by feeding adults the MEK inhibitor SL327 decreased daytime sleep and lowered waking activity. Together these data indicate ERK activation plays a role in sleep regulation (Vanderheyden, 2013).

As mentioned, inducing EGFR signaling resulted in an increase in sleep which seemed to correlate nicely with the increase in ppERK. Although these data strongly suggested that the increase in sleep was due to ppERK activation, ppERK was not directly manipulated. Thus these studies confirm and extend data demonstrating that directly activating ppERK can increase sleep. While the largest effects of sleep were obtained using a pan neuronal activation of EGFR signaling, it has also been reported that the EGFR induced increase in sleep could be mapped to the PI. Surprisingly, this study did not see any changes in sleep when ppERK was expressed in the PI using the same GAL4 drivers as a previous study. These data suggest that in the PI, EGFR activation may recruit additional factors along with ppERK to alter sleep. Such regulation may be particularly important for allowing ppERK to carry out multiple functions in various circuits as needed (Vanderheyden, 2013).

In flies, sleep homeostasis is primarily observed during the subjective day. Similarly, social enrichment also produces increases in daytime sleep. Thus, the data indicate that modulating ERK activity in the adult produces a change in sleep during the portion of the circadian day during which sleep deprivation and social enrichment modify sleep time. Interestingly, ERK activation not only increases daytime sleep, but it also results in the proliferation of terminals in the wake-promoting LNvs. The ability of ERK activation to increase synaptic terminals is reminiscent of the change in synaptic markers and structural morphology that are independently observed following sleep deprivation and social enrichment. Interestingly, arouser mutants show both enhanced ethanol sensitivity and an increase in terminals from the LNvs through its activation by EGFR/ERK. Given that a well characterized function of ERK is to regulate synaptic morphology, the current results suggest that ERK activation may be a common mechanism linking waking experience, plasticity and sleep (Vanderheyden, 2013).

As mentioned, ERK activation has been correlated with sleep time following rhomboid mediated activation of EGFR. Interestingly, in that study, ppERK was not detectable in cell bodies following rho mediated increases in sleep suggesting that, during rho activation, ppERK might be modifying sleep at the level of translation initiation. The current data extend these observations and provide genetic evidence that ERK activation may also play a role in regulating sleep and plasticity by activating gene transcription. That is, pan-neuronal expression of RSK, which retains ERK in the cytoplasm and prevents its nuclear translocation, results in a decrease in daytime sleep similar to that observed in wild type flies fed SL327. Although previous studies have established a link between CREB and sleep this study evaluated CRE-Luc solely as a reporter of transcriptional activation. The data indicates that the expression of UAS-ERKSEM increased CRE-Luc activity. In contrast, transgenic CRE-Luc reporter flies show reduced bioluminescence when crossed into a rl10a mutant background. Finally, flies fed SL327 also showed a reduction of CRE-Luc activity. These data are consistent with a recent report demonstrating that activating MEK increases bioluminescence in flies carrying a CRE-Luc reporter. Together these data suggest that ERK activation may alter plasticity and sleep, in part, by activating gene transcription (Vanderheyden, 2013).

Interestingly, expressing UAS-ERKSEM in PDF neurons does not change the number of PDF-terminals and does not alter sleep time. This is in contrast to the effects of expressing UAS-ERKSEM pan-neuronally which increases both the number of PDF-terminals and increases sleep. These data suggest that ERK activation can either influence PDF neurons in a non-cell autonomous fashion or that ERK activation is required in multiple circuits to modulate plasticity. Indeed, it has been recently shown that increasing sleep by activating the dorsal Fan Shaped Body significantly reduces the number of PDF-terminals. Thus, PDF terminal number provides an accessible read-out of brain plasticity that can be used to elucidate molecular mechanisms linking sleep and plasticity at the circuit level (Vanderheyden, 2013).

It is important to note that in flies there is a critical window of adult development that can influence sleep and learning. For example, 0-3 day old rut2080 mutants are able to respond to social enrichment with an increase in sleep but their older siblings (>3days) cannot. In other words, rut2080 mutants can exhibit higher or lower amounts of sleep as adults depending upon environmental context, not levels of rutabaga per se. Indeed, rutabaga mutants have been reported to have significant variations in sleep (both longer and shorter) compared to controls. Given that the environment can stably modify sleep during adult development, even in the absence of memory related genes, care must be taken when classifying a mutant as either long or short sleepers. It should be emphasize that the current experiments were designed to avoid making manipulations during this critical time window to avoid such confounds. However, it remains possible that ERK may modify sleep by activating additional downstream targets and/or by regulating translation initiation at the synapse (Vanderheyden, 2013).

Recent studies have shown that waking experience, including both prolonged wakefulness and exposure to enriched environments, independently produce dramatic increases in both synaptic markers and structural morphology throughout the fly brain and that these changes are reversed during sleep. To date, most studies have evaluated mutations that disrupt synaptic plasticity to identify the molecular mechanisms linking sleep with plasticity. Given that ERK is a key molecule for the regulation of synaptic plasticity and long-term potentiation, this study evaluated its ability to alter both sleep and structural plasticity. The data indicate that both sleep deprivation and social enrichment independently increase ERK phosphorylation in wild-type flies. It is also reported that expressing an active version of ERK (UAS-ERKSEM) in the adult fly results in an increase in sleep and an increase in structural plasticity in the LNvs. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep (Vanderheyden, 2013).

Dynamic MAPK signaling activity underlies a transition from growth arrest to proliferation in Drosophila scribble mutant tumors

Human tumors exhibit plasticity and evolving capacity over time. It is difficult to study the mechanisms of how tumors change over time in human patients, in particular during the early stages when a few oncogenic cells are barely detectable. This study used a Drosophila tumor model caused by loss of Scribble (Scrib), a highly conserved apicobasal cell polarity gene, to investigate the spatial-temporal dynamics of early tumorigenesis events. The fly scrib mutant tumors have been successfully used to model many aspects of tumorigenesis processes. However, it is still unknown whether the fly scrib mutant tumors exhibit plasticity and evolvability along the temporal axis. This study found that the scrib mutant tumors display different growth rates and cell cycle profiles over time, indicative of a growth arrest-to-proliferation transition as the scrib mutant tumors progress. Longitudinal bulk and single-cell transcriptomic analysis of the scrib mutant tumors revealed that the MAPK pathway, including the JNK and ERK signaling activities, shows quantitative changes over time. High JNK signaling activity causes G2/M cell cycle arrest in the early scrib mutant tumors. In addition, JNK signaling activity displays a radial polarity with the JNK(high) cells located at the periphery of the scrib mutant tumors, providing an inherent mechanism that leads to an overall JNK signaling activity decrease over time. The ERK signaling activity, in contrast to JNK activity, increases over time and promotes growth in the late-stage scrib mutant tumors. Finally, high JNK signaling activity represses ERK signaling activity in the early scrib mutant tumors. Together, these data demonstrated that dynamic MAPK signaling activity, fueled by intratumor heterogeneity derived from tissue topological differences, drives a growth arrest-to-proliferation transition in the scrib mutant tumors (Ji, 2019).

This study demonstrates that dynamic changes in JNK and ERK activities underlie a transition from a growth arrest state to a proliferation state over time in Drosophila scrib mutant tumors (Ji, 2019).

JNK signaling activation was found to exhibit a radial polarity, with JNKhigh cells located at the surface of homozygous scrib mutant tumors. The underlying reason for the heterogeneous JNK activation pattern is not yet known. One possible reason is that the active JNK ligand Eiger might be mostly provided from external sources. This might involve hemocytes or the fat body, as recently shown for another nTSG mutant alg3 . Another possible input is potential mechanical stresses that the tumor surface cells experience because JNK signaling is activated at leading-edge cells, which assemble supracellular actin cables and experience high actomyosin-dependent tensile force during dorsal closure (Ji, 2019).

JNK activation induced by injury in the wing imaginal discs also leads to cell cycle arrest at G2, which can be restored by overexpression of Cdc25/String. Expression of Cdc25/string(stg) and Cks30A anti-correlates with Mmp1 in the single-cell data. Moreover, growth arrest in early scrib mutant tumors could not be arrested by overexpression of String alone, indicating that the cell cycle defects in early scrib mutant tumors are probably mediated by multiple factors downstream of high JNK signaling activity (Ji, 2019).

Clonal scrib mutant cells are eliminated through cell competition when they are surrounded by wild-type neighbors. It is noteworthy that clonal scrib mutant cells are likely to be in a different state from growth-arrested homozygous scrib mutant cells. Studies have shown that overexpression of RasV12, NICD and p35 can effectively block the clonal scrib mutant cells from undergoing apoptosis induced by cell competition. This study found that overexpression of RasV12, NICD and p35 had little effect in relieving the early scrib mutant tumors from growth arrest. It is likely that interactions between clonal scrib mutant cells and wild-type cells during cell competition induce an additional layer of complexity into determination of clonal scrib mutant cell state, as the effects of JNK activation are known to be highly context dependent (Ji, 2019).

In human solid tumors, the tumor margin and core were also shown to experience different immune environments. This study highlights that tissue topological factors (peripheral versus center) can be an inherent source of diversity in cell populations in growing tumors and that dynamic signaling rewiring during the processes of early tumorigenesis does not necessarily require the generation of de novo mutations or new cell clones (Ji, 2019).

Signaling dynamics control cell fate in the early Drosophila embryo

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? This study combined optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. Erk activity was found to be sufficient to 'posteriorize' 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. The endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes (Johnson, 2019).

Through a combination of genetic perturbations and time-varying optogenetic stimuli, this study begins to define a model for how Erk is capable of programming three distinct cell fates the early embryo (see A conceptual model of Erk-dependent cell fate control in the early embryo). At the posterior, sustained Erk signaling induces gut endoderm, tissue that is characterized by expression of the Fog/Mist receptor-ligand pair which leads to apical constriction and tissue invagination. The boundary of this tissue is determined by the total Erk dose, and sustained Erk activity at almost any embryonic position is sufficient to trigger gut endoderm gene expression and contractility. A notable exception is the anterior pole, where the combination of Bcd and Erk switches cells to anterior fates. In the middle of the embryo, the combination of transient Erk and Dorsal normally induces the formation of ectoderm-derived neuroblasts, a fate that can be overridden by additional, ectopic Erk activity. By isolating the Erk pathway and titrating the inputs delivered, it was further shown that some cells in the embryo can adopt three distinct responses at different signaling thresholds. Lateral cells shift from no response to form intermediate neuroblasts (characterized by ind expression) or gut endoderm (marked by mist expression and contractility) as a single input parameter - the duration of Erk signaling - is increased. These three transitions are reminiscent of the requirements that define a morphogen, a substance whose concentration determines multiple distinct fates. Yet in the case of Erk it is signaling dynamics, not instantaneous concentration, which is interpreted into a cellular response (Johnson, 2019).

In contrast to the now-classic models for how Erk dynamics are decoded in cultured cells, it study findw that the early Drosophila embryo does not read the duration of a single persistent stimulus, but rather senses the cumulative load of Erk signaling. Two lines of evidence support this conclusion. First, the same overall Erk dose can be delivered in a single bolus or divided into discrete pulses spread out over a 2 h window, leading to the same effect. Second, long low-amplitude light stimuli (as well as gain-of-function mutants that activate Erk to low levels) achieve the same phenotypes as short high-amplitude light pulses. But does this distinction between duration and cumulative load sensing matter? It could be argued that it is quite important, as the putative network architectures that perform these two signal processing functions can be quite different. Persistence detection is thought to rely on network motifs like the coherent feedforward loop, whereas cumulative load detection can be implemented by combining long-term integration with an ultrasensitive downstream step. Obtaining the dynamic input-output response of a biological network can thus be a crucial first step towards a complete understanding of its network architecture and subsequent identification of molecular components. Such insights are sorely needed, as a mechanistic understanding of how signaling pathway activity is decoded into precise, reproducible patterns of gene expression is still lacking (Johnson, 2019).

There are still many unresolved questions regarding Erk-dependent cell fate choices in the early embryo. This study has shown Bicoid is sufficient to prevent posterior fates at the anterior pole, but future work must be done to dissect how Erk dynamics interact with the Bicoid gradient to pattern the formation of different anterior structures. A complete picture of anterior fate choices may benefit from precise control over both Bicoid and Erk using multi-color optogenetics. Moreover, the Erk-dependent terminal gap genes tll and hkb do much more than specify terminal fates, and participate in complex interactions with other gap and segmentation genes. Indeed, brief 15-30 min light stimuli do not affect gastrulation but lead to abdominal segment fusion, suggesting that segmentation gene network is quite sensitive to perturbations in the spatiotemporal profile of Erk signaling. The use of light to deliver quantitative spatial and temporal perturbations to gap gene expression could prove instrumental for a deeper understanding of the segmentation circuit (Johnson, 2019).

The Ras/Erk pathway is only one of many signaling outputs from receptor tyrosine kinases (RTKs), and in general it remains an open question whether light-induced Erk fully reproduces all the nuances of receptor-level stimulation. To further probe this question in the early embryo, whether OptoSOS stimulation recapitulates a classic genetic epistasis result was tested: in embryos expressing a gain-of-function Torso RTK, loss of tll can suppress the TorGOF phenotype. Indeed, this study found that the expected proportion of OptoSOS-tll treated with 45 min of light exhibit the tailless phenotype, not the TorGOF-like phenotype normally observed in OptoSOS embryos. Interestingly, suppression is lost at higher light doses, suggesting that sufficiently strong Erk activity can posteriorize embryos even in the absence of tll. Looking forward, the recent development of light-controlled RTKsopens the door to a full, systematic comparison between stimulation at the levels of the receptor versus Ras, and a quantitative comparison between these approaches is eagerly awaited (Johnson, 2019).

The relationship between Erk dynamics and cellular responses has long been studied in cultured mammalian cells. This study found that principles of dynamic control also operate to control Erk-dependent cell fates in a developing organism. It would be argued that the early Drosophila embryo is an ideal model system for dissecting dynamic control: cell fates specification occurs within 3 hours and is highly reproducible between embryos, gastrulation movements can be observed by brightfield microscopy and provide a spatially-localized readout of cell fate, the list of 'downstream' candidates for decoding dynamics is limited to a few dozen active zygotic genes in the early embryo, and the combination of optogenetic and classical genetic tools enable complex perturbations of network components. Moreover, Erk signaling in the early embryo is likely to be only one of many examples of dynamic cell fate control in vivo. The approaches outlined in this study could prove useful in many additional contexts for dissecting how developmental cell fates are specified (Johnson, 2019).


Amino Acids - 376

Structural Domains

There are several extended regions of amino acid identity with rat ERK1 and -2 and yeast FUS3 and KSS1. The sequence is most similar to rat ERK1 and -2 over its entire length (80% identity) (Biggs, 1992).

Rolled protein contains 11 conserved kinase subdomains characteristic of kinase proteins. The gain of function Sevenmaker mutation (see above: Biological overview ) is found in the C-terminal end of kinase domain XI, the putative adenosine triphospate-binding site is located in conserved domain II (Brunner, 1994a and references).

The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components (Canagarajah, 1997).

rolled/MAPK: | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 18 April 2024 
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