BIOLOGICAL OVERVIEW

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 mapk^Sem, promotes survival of extra MG. About 6.0 MG per segment survive in stage 17 mapk^Sem embryos, providing additional evidence that MAPK is required for MG survival. Remarkably, the number of surviving MG in mapk^Sem 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 mapk^Sem embryos correspond to the same MG that survive in hid mutant embryos by double mutant analysis. Stage 17 mapk^Sem; 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 mapk^Sem;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 mapk^Sem 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-hid^Ala5). The UAS-hid^Ala5 transgenes driven by sli-GAL4 induce apoptosis in the MG very efficiently. One copy of a UAS-hid^Ala5 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-hid^Ala5 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-Egfr^DN) 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, Egfr^DN 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, mapk^Sem (mapk^Sem;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 mapk^Sem embryos. Presumably, this MAPK activation in mapk^Sem 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-ERK^SEM) 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-ERK^SEM increased CRE-Luc activity. In contrast, transgenic CRE-Luc reporter flies show reduced bioluminescence when crossed into a rl^10a 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-ERK^SEM 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-ERK^SEM 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-ERK^SEM) 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).

Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo

Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. This study used 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 were found to produce transient or sustained byn expression, respectively. Although 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 (Ho, 2023).

This study has dissected the regulation of the byn stripe by combining precise optogenetic inputs in space and time with live biosensors of target gene expression. Using ectopic activation of Ras on the ventral side of wild-type embryos, high- and low-amplitude OptoSOS inputs were defined that induce distinct byn transcriptional dynamics – a pulse of expression in early NC14 versus more sustained expression – that match its endogenous responses in the posterior terminus and stripe-forming region. These conditions were then used to characterize the tll and hkb inputs that explain these byn dynamics in space and time (Ho, 2023).

This approach yielded novel insights about both the temporal and spatial interpretation of ERK inputs to pattern the byn stripe. First, differences in signal amplitude are interpreted through the timing of tll and hkb expression. The onset of tll expression is always rapid, occurring as quickly as 4 min after signaling onset, whereas there is a dose-dependent delay in the onset of hkb expression. This delay in hkb expression is a function of Ras/ERK input amplitude, not of developmental time. These data are consistent with previous observations in OptoSOS embryos that hkb RNA only accumulates to high levels in response to blue light inputs over 30 min. They also broaden the conception of the thresholds for tll and hkb expression: tll and hkb can be induced by inputs of the same amplitude, but hkb requires that the signal persist for a longer time. If the amplitude is low enough, the signal must persist longer than the developmental window allows, and hkb is never expressed. Thus, cumulative dose of ERK input (amplitude integrated over time) appears to be the relevant feature sensed by the circuit, as has been proposed for the terminal pattern as well as other systems. Integration of signal over time has similarly been shown to be important for interpretation of several morphogen pathways including Hedgehog, Wnt, Nodal and BMP. The byn circuit then processes this input through the relative timing of tll and hkb, rather than simply their presence, to determine local byn expression (Ho, 2023).

This more nuanced understanding of byn regulation resolves a conundrum of the endogenous pattern: how can the transient pulse of expression of byn in the high-ERK, Hkb-positive domain be reconciled with the presence of its inhibitor? It is shown in this study that at the high light levels which produce a comparable pulse of byn transcription, hkb transcription is delayed relative to tll and this delay is also evident in the accumulation of their protein products. Thus, there is a temporal window in which only the positive regulator is present, allowing for a pulse of byn expression, before accumulation of the repressor. The sequential appearance of Tll and Hkb was hypothesized during the initial characterizations of posterior patterning but has only now been directly shown. It is interesting to note that Tll has been characterized as a transcriptional repressor, implying that there is an intermediate node between tll and byn. However, the identity of this node and how it affects the timing of byn activation and repression remain unknown (Ho, 2023).

Improved understanding of byn regulation also explains how a byn stripe can form in conditions where tll and hkb transcription have the same spatial domain. The current study revisits these results with improved tools, in particular endogenously tagged transcriptional reporters of tll and hkb that are able to clearly resolve differences in transcriptional dynamics that were obscured by enhancer-based reporter constructs.It was found that stimulus conditions that support sustained byn can also support hkb expression in NC14, but under these conditions hkb expression is largely absent from earlier nuclear cycles. The co-expression of sustained byn with hkb under low light differs from the wild-type pattern, where the byn stripe forms in a region only expressing tll. Presumably the endogenous ERK gradient induces tll expression at even lower activity levels than optogenetic inputs. It is noted that the shortened bursting duration of sustained byn at the ectopic position (~25 min) compared with the endogenous stripe (~45 min) suggests that the late-appearing hkb under low light does ultimately repress byn in late NC14. It is also possible that the network dynamics reported in this study provide robustness to the byn circuit, allowing it to produce different outputs for even a narrow range of input strengths (Ho, 2023).

This study reveals that the tll-hkb-byn circuit can be classified as an incoherent feedforward loop with rapid activation and delayed repression, a circuit with well-characterized pulse-generation and stripe-forming properties. A unique feature of this circuit however is that the delay in hkb expression is dose-dependent, meaning that differences in signal amplitude are converted to differences in hkb dynamics and thus different byn responses (i.e. transient if hkb onset is fast, sustained if hkb onset is slow). Interestingly, similar dose-dependent delays in transcriptional onset were recently shown for Dorsal and BMP signaling targets. What is the mechanism underlying this delay in hkb onset? The dose-dependence of tll and hkb has been a longstanding open question even without the complexity of temporal dynamics. ERK signaling activates transcription of both tll and hkb through relief of the same repressor, Cic, and it is unclear why these genes would require different doses of ERK signaling. The experiments rule out a few possible explanations. Developmental time does not appear to be crucial, given that the delay in hkb transcription is observed regardless of when light is applied and both the tll and hkb loci are known to be accessible early. It is also possible to rule out interactions with other components of the anterior-posterior patterning machinery given that this study was able to produce an ectopic byn stripe rotated 90° from its endogenous counterpart. One intriguing possibility, supported by previous ChIP-seq results, is that Cic leaves the enhancers of hkb more slowly than those of tll. It is also possible that signaling-dependent chromatin changes are involved. These models will be tested in future studies (Ho, 2023).

The second major finding is that the boundary of a uniform OptoSOS input is blurred in space downstream of Ras to produce two domains from a single input – a transient byn domain within the high-ERK illuminated region and a sustained domain in the low-ERK unilluminated region. These non-local effects of a local Ras input are most likely mediated by diffusion of active intracellular components, a well-established contributor to developmental patterning in the syncytial Drosophila embryo. It remains unknown whether the endogenous terminal dpERK gradient is produced from a similar gradient of active Torso receptors, or is due to the combination of a discrete domain of Torso activity at the poles and cytoplasmic diffusion of downstream components. If the latter model is correct, the developmental rescue by an all-or-none OptoSOS input may not be an example of a simple input replacing the function of a complex one, but rather a good approximation of endogenous activation in the terminal system. A number of systems once thought to depend strictly on input concentration have similarly been shown to depend on an unexpectedly simple form of the input (Ho, 2023).

Several limitations of the optogenetic system reveal opportunities for future investigation. These experiments were performed at an ectopic position in the embryo where position-specific gene expression may influence ERK interpretation differently than at the poles. For example, the gap gene knirps has been shown to repress tll in the center of the embryo, and it was observed that the total domain of tll and byn expression was smaller under low light. Because of these positional differences in ERK sensitivity, it is not possible to make absolute comparisons about input and output strengths with the endogenous terminal pattern. In the future it will be interesting to investigate this circuit in embryos lacking other sources of positional information, preventing localized gap gene expression . Also, it is possible that the methods left some transcriptional bursts undetected, and it is not possible to distinguish whether an upper bound of ~75% transcriptionally active nuclei represents true transcriptional heterogeneity or an experimental limit of detection. These limitations could be overcome by future studies using techniques that simultaneously label the target DNA locus and measure transcription in live embryos, or advances in high-quality volumetric imaging and machine-learning approaches. Finally, many questions remain about the precise temporal relationships between ERK activation, gene transcription and protein accumulation. What is the relative influence of tll and hkb transcripts produced by early versus late nuclear cycles, and what is the delay between RNA production and protein accumulation? Combining transcriptional and protein reporters in the same embryo with mathematical models will allow these questions to be addressed (Ho, 2023).

Altogether, this work provides a blueprint for dissecting a developmental circuit with optogenetic tools to reveal new insights about network architecture. This study has manipulated amplitude, duration, timing and spatial pattern of the signal to understand the contributions of each factor to signal interpretation. This framework will be an effective strategy for dissecting other developmental circuits in the future (Ho, 2023).

PROTEIN STRUCTURE

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).

date revised: 18 June 2024 

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