InteractiveFly: GeneBrief raw: Biological Overview | References


Gene name - raw

Synonyms -

Cytological map position - 29E4-29E6

Function - signaling

Keywords - dorsal open group, JNK/AP-1 pathway antagonist

Symbol - raw

FlyBase ID: FBgn0003209

Genetic map position - 2L:8,709,870..8,740,633

Classification - novel non-conserved sequence

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Zhou, J., Edgar, B. A. and Boutros, M. (2017). ATF3 acts as a rheostat to control JNK signalling during intestinal regeneration. Nat Commun 8: 14289. PubMed ID: 28272390
Summary:
Epithelial barrier function is maintained by coordination of cell proliferation and cell loss, whereas barrier dysfunction can lead to disease and organismal death. JNK signalling is a conserved stress signalling pathway activated by bacterial infection and tissue damage, often leading to apoptotic cell death and compensatory cell proliferation. This study shows that the stress inducible transcription factor ATF3 restricts JNK activity in the Drosophila midgut. ATF3 regulates JNK-dependent apoptosis and regeneration through the transcriptional regulation of the JNK antagonist, Raw. Enterocyte-specific ATF3 inactivation increases JNK activity and sensitivity to infection, a phenotype that can be rescued by Raw overexpression or JNK suppression. ATF3 depletion enhances intestinal regeneration triggered by infection, but does not compensate for the loss of enterocytes and ATF3-depleted flies succumb to infection due to intestinal barrier dysfunction. In sum, this study has provided a mechanism to explain how an ATF3-Raw module controls JNK signalling to maintain normal intestinal barrier function during acute infection.
Luong, D., Perez, L. and Jemc, J. C. (2018). Identification of raw as a regulator of glial development. PLoS One 13(5): e0198161. PubMed ID: 29813126
Summary:
Glial cells perform numerous functions to support neuron development and function, including axon wrapping, formation of the blood brain barrier, and enhancement of synaptic transmission. A novel gene, raw, has been identified that functions in glia of the central and peripheral nervous systems in Drosophila. Reducing Raw levels in glia results in morphological defects in the brain and ventral nerve cord, as well as defects in neuron function, as revealed by decreased locomotion in crawling assays. Examination of the number of glia along peripheral nerves reveals a reduction in glial number upon raw knockdown. The reduced number of glia along peripheral nerves occurs as a result of decreased glial proliferation. As Raw has been shown to negatively regulate Jun N-terminal kinase (JNK) signaling in other developmental contexts, the expression of a JNK reporter and the downstream JNK target, matrix metalloproteinase 1 (mmp1) was examined, and raw knockdown was found to result in increased reporter activity and Mmp1 levels. These results are consistent with previous studies showing increased Mmp levels lead to nerve cord defects similar to those observed upon raw knockdown. In addition, knockdown of puckered, a negative feedback regulator of JNK signaling, also causes a decrease in glial number. Thus, these studies have resulted in the identification of a new regulator of gliogenesis, and demonstrate that increased JNK signaling negatively impacts glial development.
BIOLOGICAL OVERVIEW

High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. This study shows that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. These studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, evidence is provided that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor (Bates, 2008).

The initial molecular and genetic studies of the dorsal-open mutant raw revealed it to encode a widely expressed and novel gene product, required for the restriction of JNK/AP-1 activity to LE epidermal cells (Byars, 1999). The Raw protein sequence yielded no insights into its mechanism of function as the Raw sequence harbors none of the canonical motifs that are associated with nuclear localization, phosphorylation, membrane insertion, or protein secretion. Mechanistic studies of a novel protein can be challenging, but this study reports use of a variety of genetic strategies to probe Raw function and test models of AP-1 silencing. In particular (1) the epistatic relationship of raw to genes encoding well-characterized JNK-signaling components was assessed, (2) genes, which have designated the raw group, have been assessed that share an array of loss-of-function phenotypes, (3) the interaction phenotypes among the raw-group loci were determined, and (4) raw transgenics were generated, that were utilized to probe sites of Raw function. These analyses reveal that raw belongs to a small set of dorsal-open group genes that encode JNK/AP-1 pathway antagonists. The characterization of raw, and the raw group more generally, has led to a new appreciation of wide-ranging competence for AP-1 activity in early Drosophila embryos. As signal activation is critical for proper development, so also is its silencing (Bates, 2008).

The current study shows that although raw functions upstream of Jra as an AP-1 antagonist, its action is independent of the bsk-encoded kinase that is required to activate AP-1 activity in LE cells during closure. In addition, raw is required broadly in the epidermis to effect normal dorsal closure. Overall, these studies expose the importance of epidermal AP-1 silencing during embryogenesis and lead to an extension of existing models for dorsal closure, which have largely confined their focus to mechanisms of JNK/AP-1 activation in LE cells. In particular, the data indicate that Raw and the other raw-group gene products (Puckered and Ribbon) function to silence Basket JNK-independent AP-1 activity in the embryonic dorsolateral epidermis. AP-1 silencing, via the combined actions of the raw-group gene products, essentially wipes the epidermal slate clean and primes the system for activation via a still unidentified deterministic signal that acts only in LE cells (Bates, 2008).

The AP-1 abnormality in raw-group mutant embryos has not yet been molecularly defined. Previous studies provide compelling evidence that AP-1 overexpression in Drosophila embryos is not sufficient to disrupt either dorsal closure or development more generally. It seems unlikely, therefore, that elevated levels of the AP-1 transcription factor in raw-group mutants simply override a requirement for kinase activation in initiating an AP-1-dependent program of gene expression. Instead, it is speculated that AP-1 is aberrantly modified in raw-group mutant embryos. It might be that AP-1 escapes inactivation in mutants; either alternatively or additionally, AP-1 in mutants may be inappropriately activated via phosphorylation. In addition to Basket JNK, there are four other Drosophila MAP Kinases (p38a, p38b, Mpk2, and Rolled) that might provide dysregulated kinase activity in mutants. Consistent with this idea is the observation that the oogenesis phenotypes associated with raw (and puc) ectopic expression and mutation have considerable similarity with gain- and loss-of-function phenotypes associated with mutations in the p38 pathway that is required in the germ line for proper oogenesis. Finally, a kinase-dependent activation model for epidermal Jun provides the most parsimonious explanation for ectopic epidermal signaling observed in puc MPK-deficient embryos. From the perspective of regulated signaling more generally, however, lowering an AP-1 activity baseline in wild-type embryos will (1) provide a means for the clean on/off regulation of JNK/AP-1 that has been predicted in computer simulations and (2) make a less strenuous demand on the input activating signal (Bates, 2008).

The discovery that null alleles of raw and puc interact, with double mutants exhibiting an embryonic lethal phenotype distinct from their shared loss-of-function null phenotypes, revealed the independent contributions of raw and puc to embryogenesis, presumably through their effects on AP-1 antagonism. Drosophila overexpression studies have previously implicated several pathways in the parallel control of AP-1 activity, but this analysis represents the first direct demonstration of physiologically relevant, parallel regulatory pathways (Bates, 2008).

The genetic interaction that was documented between null alleles of raw and puc contrasts with the lack of a detectable interaction between null alleles of raw and rib. Moreover, the observation that raw and rib hypomorphs interact genetically during dorsal closure is consistent with previously published data, as well as with findings documenting (1) raw/rib interactions in several other epithelial tissues, including the nervous system, salivary gland, trachea, and gut (Blake, 1998; Blake, 1999) and (2) overlapping raw and rib expression patterns in Drosophila embryos (Byars, 1999). Together, results from these genetic and molecular studies point to roles for raw and rib in a single, previously unrecognized puc-independent AP-1 inactivation system (Bates, 2008).

In addition to providing evidence for raw-mediated global silencing of AP-1, this study underscores a simultaneous requirement for a biologically appropriate activator of JNK/AP-1 signaling. In this regard, expression of raw in LE cells failed to rescue raw-dependent defects in dorsal closure. Even more notable, however, was the observation that overexpression of raw+ in wild-type embryos, and in wild-type LE cells in particular, had no detrimental effects on embryonic development and dorsal closure. From a signaling perspective this result indicates that JNK-dependent AP-1 can be activated despite expression of the wild-type raw gene product, and thus Raw does not function as a binary switch for signaling. Although it is formally possible that LE expression of raw was initiated too late to disrupt JNK/AP-1 signaling and dorsal closure in the LE-gal4/UAS-raw+ transgenics, this interpretation is not favored since the LE-GAL4 driver used in this study has been shown previously to (1) be an effective driver of at least one gene that is required in LE epidermal cells for closure and (2) drive expression of a lacZ reporter in LE cells during dorsal closure (Bates, 2008).

The finding that raw expression in LE cells is not sufficient to inactivate AP-1 activity in a cell-autonomous fashion is consistent with models for independent, developmentally regulated triggers of JNK signaling. Indeed, there is abundant experimental support for developmentally regulated activation of JNK signaling in LE cells. JNK/AP-1 activation likely follows an amalgamation of signals, both from the amnioserosa and the epidermis, both in the form of cytoskeletal components and signaling molecules. Among the best candidates with postulated roles in JNK/AP-1 activation are small GTPases, nonreceptor tyrosine kinases, and integrins. Thus, despite the broad epidermal competence for AP-1 signaling that has been shown in this work, the activation signal is itself limited to only LE cells and functions via an unknown mechanism. Importantly, AP-1 antagonism by raw cannot override its signal-dependent activation in the LE (Bates, 2008).

dpp, when expressed pan-epidermally, leads to a raw-like phenotype: embryonic lethality associated with ventral cuticular defects. In a direct assessment of equivalence of raw loss-of-function and dpp gain-of-function ventral cuticular phenotypes, whether pan-epidermal expression of brinker (brk) can rescue raw-dependent defects in the ventral cuticle was tested. The Dpp signaling modifier Brinker functions by negatively regulating dpp target genes (Bates, 2008).

This study found that although brk is normally expressed in nonoverlapping lateral and ventral domains of the embryonic epidermis, it is undetectable in the epidermis of embryos homozygous for a null allele of raw. It was also found that although brk+ fails to rescue raw-dependent defects in dorsal closure, it does rescue raw-dependent defects in the ventral cuticle. Together, these data point to an important role for dpp, brk, and/or their target genes in development of the ventral epidermis (Bates, 2008).

What cannot be discerned from these studies is (1) how the nonoverlapping epidermal domains of dpp and brk are established and maintained and (2) if and how epidermal dpp and brk interact during normal embryonic development. In this regard, a previous finding that LE dpp is not autoregulatory makes it unlikely that brk functions in direct fashion to set the LE dpp expression boundary. Even more significant is the finding that cuticles derived from Jra raw double mutants exhibit defects in dorsal closure, but not ventral cuticular patterning (Byars, 1999). Indeed, these data highlight the requirement for functional Jun in generating ventral cuticular defects in raw mutant embryos. Taken together then, these data suggest that the effects of JNK/AP-1-activated dpp in the dorsal epidermis of raw mutant embryos are far reaching, extending even to the most ventral regions of the embryo (Bates, 2008).

Having established a dependence upon Jun for raw-dependent ventral cuticular defects, it is postulated that the absence of brk in raw mutant embryos is a direct consequence of ectopic JNK/AP-1 activity in the dorsal epidermis of these mutants. It is suspected that ectopic JNK/AP-1 activity leads secondarily to ectopic dpp activity, and that in its turn ectopic dpp activity leads finally to brk repression. An alternative view, that raw might have dual regulatory roles in the epidermis, seems less likely although it is not absolutely excluded by this strictly genetic analysis. In this regard, in addition to its function as a JNK/AP-1 antagonist in the embryonic dorsal epidermis, raw might function independently as a trigger of brk expression in the ventral epidermis. Clearly, the mechanism of raw function and the relationship of dpp to brk in eliciting properly formed ventral cuticle warrant further investigation (Bates, 2008).

In Drosophila, as in all animals, signaling pathways are finely regulated at several levels. Although there are multiple tiers of regulation operating on the JNK/AP-1 signaling cascade, surprisingly little of the regulation of this pathway is known. This study of the functions and interactions of a subset of dorsal-open group genes (raw, rib, and puc) has shed some additional light on both old (puc-mediated) and new (raw/rib-mediated) mechanisms of JNK/AP-1 antagonism. These data indicate that Raw functions to silence Basket JNK-independent AP-1-mediated transcription and to set the stage for JNK-dependent regulation of transcription. The suggestion that spatial restriction of the JNK/AP-1 signal requires antagonists, as well as activators, is not without precedent in other signaling systems. Many signaling pathways have already been shown to be multilayered and to depend heavily on negative regulation to terminate developmental events, and/or control both the distance and speed that a signal can move (e.g., Nodal). In addition, and as was suggested is the case for the Drosophila JNK/AP-1 pathway, reducing basal levels of a signaling pathway can augment the effects of its signaling responses (e.g., Hedgehog and Lef1) (Bates, 2008).

Finally, given the numerous associations of improper JNK/AP-1 activity with human disease, it seems apparent that many cell types have the capacity to signal via the JNK/AP-1 pathway. Presumably, this capacity is diminished (and then tightly regulated) during normal vertebrate development and aging. Viewed from this perspective, characterization of Raw as an essential AP-1 antagonist establishes a clear basis for future studies of AP-1 regulation (Bates, 2008).

Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw

The directional flow of information in neurons depends on compartmentalization: dendrites receive inputs whereas axons transmit them. Axons and dendrites likewise contain structurally and functionally distinct subcompartments. Axon/dendrite compartmentalization can be attributed to neuronal polarization, but the developmental origin of subcompartments in axons and dendrites is less well understood. To identify the developmental bases for compartment-specific patterning in dendrites, a screen was carried out for mutations that affect discrete dendritic domains in Drosophila sensory neurons. From this screen, mutations were identified that affected distinct aspects of terminal dendrite development with little or no effect on major dendrite patterning. Mutation of one gene, raw, affected multiple aspects of terminal dendrite patterning, suggesting that Raw might coordinate multiple signaling pathways to shape terminal dendrite growth. Consistent with this notion, Raw localizes to branch-points and promotes dendrite stabilization together with the Tricornered (Trc) kinase via effects on cell adhesion. Raw independently influences terminal dendrite elongation through a mechanism that involves modulation of the cytoskeleton, and this pathway is likely to involve the RNA-binding protein Argonaute 1 (AGO1), as raw and AGO1 genetically interact to promote terminal dendrite growth but not adhesion. Thus, Raw defines a potential point of convergence in distinct pathways shaping terminal dendrite patterning (Lee, 2015).

Although the concept of positional information was first applied to embryonic development, intracellular positional information governs morphogenesis of individual cells as well. For example, positioning the nucleus at the cell center and growth zones at the cell periphery depends on positional information from the microtubule cytoskeleton in Schizosaccharomyces pombe. Several lines of evidence support the existence of distinct subcompartments in axons and dendrites, but the forms of intracellular positional information and the coordinate systems that guide the development of these subcompartments have not been extensively characterized. Results from this screen and other studies suggest that at least two types of positional information govern C4da dendrite patterning. First, terminal branch distribution along the proximal-distal axis depends on microtubule-based processes; perturbing microtubule-based transport leads to a distal-proximal shift in the distribution of terminal dendrites in C4da arbors. Interestingly, modulating the activity of the F-actin nucleator Spire also affects terminal dendrite positioning along the proximal-distal axis, suggesting that multiple pathways contribute to the fidelity of branch placement. Second, terminal dendrites rely on dedicated programs that may act locally to regulate terminal dendrite patterning. The observation that different pathways regulate different aspects of terminal dendrite development suggests that multiple signaling systems exist for the local control of dendrite growth (Lee, 2015).

This study identified raw as a key regulator of terminal dendrite patterning. raw encodes a membrane protein that accumulates at branch-points and coordinately regulates terminal dendrite adhesion/stability via a pathway that involves Trc and terminal dendrite elongation via a pathway that is likely to involve cytoskeletal remodeling and AGO1. Raw therefore provides a potential point of integration for external signals that regulate these downstream growth programs. These pathways could be responsive to the same signal -- for example, Raw association with an extracellular ligand or a co-receptor -- or could be spatially/sequentially segregated. Identification of additional raw-interacting genes should help clarify the architecture of these signaling pathways (Lee, 2015).

Raw regulates cell-cell signaling, and in gonad morphogenesis Raw modulates Cadherin-based interactions between somatic gonadal precursor cells and germ cells, in part by localizing Armadillo to the cell surface. Likewise, the data support a role for Raw in promoting Trc activation by localizing Trc to the plasma membrane. Thus, one plausible model for Raw function in dendrite development is that it interacts with an extracellular signal, which might be a component of the ECM or a cell surface protein on epithelial cells, and signals together with a co-receptor to stimulate downstream pathways for adhesion and cytoskeletal remodeling. Several analogous signaling systems involving interactions with the epidermis that influence terminal dendrite or sensory axon patterning have been described, but how many of these signaling systems are at work in a given neuron, and how Raw interfaces with other signaling pathways, remain to be determined (Lee, 2015).

Although Raw has no obvious vertebrate counterpart, stretches of the ECD bear similarity to mucins and leucine-rich repeat proteins, one of which might serve an analogous function. Moreover, components of both downstream signaling pathways that this study identified are conserved in vertebrates and play known roles in dendrite patterning, including roles in the local control of dendrite growth: the Trc orthologs NDR1/2 (STK38/STK38L) regulate aspects of dendrite branch and spine morphogenesis, and Argonaute proteins mediate miRNA-mediated control of dendrite patterning, in part through local effects on translation. Additionally, dendrites contain structures related to P-granules, and Argonaute proteins may influence local translation in P-granules as well. Thus, versions of the Raw-regulated signaling pathways might control terminal dendrite patterning in vertebrates (Lee, 2015).

raw Functions through JNK signaling and cadherin-based adhesion to regulate Drosophila gonad morphogenesis

To form a gonad, germ cells (GCs) and somatic gonadal precursor cells (SGPs) must migrate to the correct location in the developing embryo and establish the cell-cell interactions necessary to create proper gonad architecture. During gonad morphogenesis, SGPs send out cellular extensions to ensheath the individual GCs and promote their development. Mutations have been identified in the raw gene that result in a failure of the SGPs to ensheath the GCs, leading to defects in GC development. Using genetic analysis and gene expression studies, it was found that Raw negatively regulates JNK signaling during gonad morphogenesis, and increased JNK signaling is sufficient to cause ensheathment defects. In particular, Raw functions upstream of the Drosophila Jun-related transcription factor to regulate its subcellular localization. Since JNK signaling regulates cell adhesion during the morphogenesis of many tissues, the relationship was examined between raw and the genes encoding Drosophila E-cadherin and beta-catenin, which function together in cell adhesion. It was found that loss of DE-cadherin strongly enhances the raw mutant gonad phenotype, while increasing DE-cadherin function rescues this phenotype. Further, loss of raw results in mislocalization of beta-catenin away from the cell surface. Therefore, cadherin-based cell adhesion, likely at the level of beta-catenin, is a primary mechanism by which Raw regulates germline-soma interaction (Jemc, 2012).

raw is an important regulator of embryonic gonad morphogenesis and the establishment of proper gonad architecture. raw mutants exhibit a failure of SGPs to ensheath GCs in the gonad, resulting in defects in GC development. It was also found that raw affects gonad morphogenesis primarily by acting as a negative regulator of the JNK signaling pathway. Finally, it was found that raw mutants exhibit defects in cadherin-based cell adhesion, and that this is the primary cause of the failure of gonad morphogenesis. These results have clear implications for understanding of how important cell signaling pathways are regulated to control normal organogenesis and may be misregulated to cause disease (Jemc, 2012).

Previously raw has been proposed to be a negative regulator of the JNK pathway during closure of the dorsal epidermis, based on changes in JNK-dependent expression of target genes such as dpp and puc. Indeed, an increase was seen in puc expression in the region of the embryonic gonad, and more broadly throughout the embryo. Further, upregulation of a dedicated AP-1 reporter construct was observed, indicating that the changes in target gene expression are directly due to changes in AP-1 transcriptional activity regulated by the JNK pathway. When the JNK pathway was upregulated via independent means, similar defects were observed in gonad morphogenesis, indicating that the changes in the JNK pathway were the primary mechanism by which raw mutants cause gonad defects. Therefore, the results support and extend the previous observations that raw acts as a negative regulator of JNK pathway, both in the gonad and in other tissues in which raw mutants exhibit defects in morphogenesis (Jemc, 2012).

How might raw be regulating the JNK pathway? The evidence indicates that raw regulates the JNK pathway at the level of transcription factor JRA. It was found that the nuclear localization of JRA, but not FOS, was altered in raw mutants. JRA was more strongly concentrated in the nucleus in a variety of cell types in raw mutants, whereas no changes were observed in the global levels of JRA protein. These observations are consistent with previous genetic epistasis experiments that indicated that raw acts at the level of JRA, rather than further upstream in the pathway. It has been proposed that raw acts as a general negative regulator of the JNK pathway to suppress basal activity and perhaps establish a threshold for pathway activation. The data are consistent with this hypothesis, as a general nuclear accumulation of JRA was seen in a variety of cell types in the embryo, along with generalized activation of the transcriptional reporter for AP-1 activity. Presumably, not all of these different cells are normally exposed to activators of the JNK pathway at this time, indicating that the pathway may be activated in cells in which the pathway would normally be turned off. Thus, rather than being just a modulator of the level of signal a cell might receive under conditions of JNK pathway activation, raw is likely also responsible for ensuring that the pathway remains inactive in cells that are not experiencing pathway activation (Jemc, 2012).

It is difficult to predict exactly how Raw may be regulating JNK signaling, as the Raw protein has no readily identifiable protein domains and exhibits only limited homology to proteins of other species. It may be the case that similar JNK pathway regulators are present in other species and have structural and/or functional conservation with Raw, but are difficult to identify based on primary sequence homology. Studies examining the subcellular localization of Raw in cultured mammalian or Drosophila cells indicate that it is primarily found in the cytoplasm. One attractive hypothesis is that Raw directly binds to JRA to block its nuclear translocation and sequester JRA in the cytoplasm. Unfortunately, efforts to identify a direct, physical interaction between Raw and JRA have so far been unsuccessful (Jemc, 2012).

The JNK pathway is subject to negative regulation at several levels. Most familiar are the MAP kinase phosphatases (MKPs, a subfamily of Dual-specificity phosphatases), like Drosophila Puckered, that provide negative feedback by dephosphorylating activated MAP kinases such as JNK. Additional modes of regulation include nuclear repressors of AP-1 target genes (e.g., Anterior open) and a secreted protease that acts in negative feedback on the JNK pathway (Scarface). Raw appears to represent a distinct mode of regulation, acting on the ability of JRA/JUN to translocate to the nucleus. Regulation of the subcellular localization of transcription factors and cofactors is a strategy that is commonly deployed to regulate signaling pathway activity, and many transcription factors are sequestered in the cytoplasm as a mechanism for negatively regulating their activity. It is proposed that JRA is subject to such regulation as a means to repress its activity in cells that are not experiencing sufficient levels of JNK pathway activity (Jemc, 2012).

Further studies of Raw are necessary to determine how Raw functions at a molecular level to regulate JRA subcellular localization (Jemc, 2012).

This study has found that raw mutants also exhibit defects in cadherin-based cell adhesion, which is known to be important for proper gonad morphogenesis and GC ensheathment by SGPs. Loss of DE-cad function exacerbates the gonad defects observed in raw mutants while increasing DE-cad function strikingly rescues these defects. It is likely that the increase in JNK pathway activity in raw mutants leads to defects in DE-cad-based adhesion and that this is the primary cause of the gonad morphogenesis defects. This is in contrast to the role of raw and the JNK pathway in the closure of the dorsal epidermis, which is largely thought to be due to regulation of dpp expression. Consistent with this, less up-regulation of dpp was observed in the region of the gonad, relative to the overall activation of the AP-1 transcriptional reporter (Jemc, 2012).

Previous studies in mammalian cells have implicated JNK signaling in negative regulation of cadherin-based cell adhesion, while in other contexts the JNK pathway has also been observed to upregulate DE-cad. The current results favor a repressive role for the JNK pathway on DE-cad in the gonad. It is also known that cadherins can act upstream of the JNK pathway, and that loss of cadherin can lead to an increase in c-Jun protein levels. However, the current results are consistent with DE-cad acting downstream of the JNK pathway, since DE-cadherin expression could rescue gonad morphogenesis independently of rescuing JRA localization. It is concluded that during gonad morphogenesis, raw acts as a negative regulator of the JNK pathway, and increased JNK pathway activity observed in raw mutants leads to a downregulation of DE-cadherin based cell adhesion and a failure of proper ensheathment of the GCs by the somatic gonad (Jemc, 2012).

While no change was observed in DE-cad localization in the gonad, the localization of ARM/ß-catenin was dramatically altered. Since ARM is essential for proper DE-cad function in cell adhesion, this indicates that DE-cadherin-based adhesion is strongly affected in raw mutants. It has been shown that JNK can directly phosphorylate ß-catenin and negatively regulate its activity. Consistent with this, a modest increase was observed in the relevant phospho-form of ARM/ß-catenin in raw mutants. Thus, this may represent one aspect of how the JNK pathway regulates DE-cad based adhesion in the gonad. However, the change in ARM/ß-catenin phosphorylation observed is unlikely to account for the more dramatic change in ARM/ß-catenin immunostaining observed in the gonad. Considering that a strong increase was also observed in transcriptional activation by AP-1 in raw mutants, and that mutations in the JRA transcription factor can partially suppress the gonad morphogenesis defects observed in raw mutants, it is concluded that at least some of the JNK pathway effect on DE-cad function and ß-catenin localization is likely to depend on changes in gene expression mediated by AP-1. Since no overall changes were observed in protein levels for DE-cad or ARM/ß-catenin in raw mutants, the changes in gene expression may reflect changes in other regulators of DE-cad based cell adhesion. Interestingly, previous work identified a zinc transporter, Fear of intimacy, that also affects gonad morphogenesis and GC ensheathment by regulating DE-cad. Regardless of whether there is an interesting connection between zinc transport and the JNK pathway, or these represent independent pathways, they highlight the importance of careful regulation of cadherin-based cell adhesion in controlling morphogenesis (Jemc, 2012).

Previous work has indicated that GC ensheathment requires preferential adhesion between SGPs and GCs, such that SGP-GC adhesion is favored over GC-GC or SGP-SGP adhesion. Indeed, just increasing the adhesion between GCs via DE-cadherin expression in these cells is sufficient to prevent ensheathment of the GCs by SGPs. In raw mutants, changes were primarily observed in the JNK pathway in SGPs and surrounding somatic cells. In addition, expression of DE-cad in the soma, but not the germline, is sufficient to rescue the ensheathment defects in raw mutants. Together, these data indicate that raw mutants likely affect gonad ensheathment by decreasing DE-cad function in the SGPs, which decreases SGP-GC adhesion relative to GC-GC adhesion. While it is possible that effects of raw on somatic cells outside of the gonad affect ensheathment within the gonad, it is less easy to imagine how decreasing DE-cad activity in these cells would influence ensheathment (Jemc, 2012).

The JNK pathway has been implicated in many diseases, including birth defects, neurodegeneration, inflammatory diseases, and cancer. Signaling pathways must be tightly regulated both positively, to ensure rapid and robust signaling responses, and negatively, to terminate signaling events and prevent inappropriate signaling. As a negative regulator of JNK pathway signaling, raw represents the type of gene that might be mutated or misregulated in diseases caused by altered JNK pathway activity. This idea is supported by the strong developmental phenotypes associated with mutations in negative regulators of the JNK pathway in Drosophila and mice (Jemc, 2012).

One disease where the JNK pathway has been particularly well studied is cancer. The JNK pathway's role in cancer is complex, however, and the pathway can act in tumor suppression or oncogenesis, depending on the context. In mouse and Drosophila models of cancer due to activated Ras, upregulation of the JNK pathway is required for tumor formation and disease progression. Interestingly, downregulation of E-cadherin is also associated with cancer progression, including in the models of activated Ras where the JNK pathway is involved. Thus, a similar link between the JNK pathway and cadherin regulation that was observed in morphogenesis of the gonad during development may play a role in oncogenesis. Since upregulation of the JNK pathway promotes cancer in these examples, negative regulators of the pathway such as the MAPK phosphatases or Raw would act as tumor suppressors whose mutation could contribute to disease progression. A better understanding of how the JNK pathway is regulated, and how Raw contributes to this regulation, is essential for understanding the normal roles of the JNK pathway in development and homeostasis, and how it is misregulated to cause disease (Jemc, 2012).

The dorsal-open group gene raw is required for restricted DJNK signaling during closure

During dorsal closure in Drosophila melanogaster, cells of the lateral epidermis migrate over the amnioserosa to encase the embryo. At least three classes of dorsal-open group gene products are necessary for this morphogenetic movement. Class I genes code for structural proteins that effect changes in epidermal cell shape and motility, including zipper, coracle, canoe and myospheroid. Class II and III genes code for regulatory components of closure: Class II genes encode Drosophila Jun amino (N)-terminal kinase (DJNK) signaling molecules, including misshapen, hemipterous, basket, Jun-related antigen, kayak, anterior open/yan and puckered, and Class III genes encode Decapentaplegic-mediated signaling molecules. All characterized dorsal-open group gene products function in the epidermis. Reported here is a molecular and genetic characterization of raw, a newly defined member of the Class II dorsal-open group genes. The novel protein encoded by raw is required for restriction of DJNK signaling to leading edge epidermal cells as well as for proper development of the amnioserosa. Taken together, these results demonstrate a role for Raw in restriction of epidermal signaling during closure and suggest that this effect may be mediated via the amnioserosa (Byars, 1999).

The distribution of raw transcripts, which is uniform in early embryos, becomes more refined as development proceeds. In gastrulating embryos, raw transcripts are evident in the midgut rudiments as well as in the ventral furrow and cephalic and dorsal folds. In germband-extended-stage embryos, raw transcripts appear limited to the gut primordia and ventral neurogenic region. This spatial restriction continues in germband-retracted-stage embryos where transcription is most conspicuous in the central nervous system (CNS) and midgut. Although raw is transcribed throughout the CNS in later stages, its pattern of expression is refined in the midgut. Here, raw transcription is limited primarily to the second midgut constriction. The patterns of raw transcription observed correlate well with documented roles for raw in the nervous system and intestinal tract; however, they provide little insight into how Raw might affect dorsal closure. All previously characterized dorsal-open group genes are expressed in the epidermis or in the epidermis and amnioserosa, and raw could not be detected in either of these tissues. Reports that amnioserosal gene transcription is sometimes recalcitrant to staining in standard in situ staining protocols led the authors to evaluate raw expression by other means. More notably, reporter expression allowed for detection of beta-galactosidase in the amnioserosa (Byars, 1999).

Raw function could affect dorsal closure at any of several steps in the morphogenetic pathway. It could play a structural role in either the amnioserosa or the epidermis. As examples, Raw might be required for the physical interaction of the opposing tissues during closure or for the restructuring of the cytoskeleton that is associated with changes in epidermal cell shape during closure. Alternatively, Raw could play a signaling role, providing either an amnioserosal or epidermal cue that regulates signaling in the epidermis during closure. In an effort to distinguish between these structural and instructive models and to understand better the role of Raw in dorsal closure, epidermal morphology and signaling was examined Epidermal cell elongation, a hallmark of dorsal closure, is thought to be driven by the movement of a myosin motor with a filamentous actin (F-actin) substrate in leading edge cells. Several lines of evidence indicate that this initiating event of closure occurs normally in raw mutant embryos. Proper F-actin localization has been documented in raw mutant embryos by staining with phalloidin. The fact that F-actin accumulates normally in leading edge epidermal cells and that these cells can elongate in animals harboring either hypomorphic or amorphic raw alleles indicates that dorsal closure initiates properly in raw mutants. Moreover, these data suggest that the DJNK signaling pathway is operative in the epidermal leading edge of raw mutant embryos (Byars, 1999).

To more directly test DJNK activation in raw mutants, expression of dpp and puc, the two known transcriptionally regulated targets of DJNK signaling during closure, were examined. In wild-type embryos, epidermal expression of both dpp and puc is dependent upon DJNK signaling and is confined to leading edge cells. Transcription of these targets is abolished in leading edge epidermal cells in hep (DJNKK), bsk (DJNK) and Jra (DJun) mutant embryos, and expanded in embryos overexpressing either activated c-Jun or wild-type DJun. As seen in embryos with ectopic DJun function, the domains of dpp and puc transcription in the epidermis of raw mutant embryos are markedly expanded beyond their normal ranges. It was also noted that puc transcription, as assayed by an enhancer reporter, expands to a greater lateral distance in raw mutants than in puc mutants. These data demonstrate that Raw is required for restriction of dpp and puc to the leading edge of the dorsal epidermis, and point to an upstream role for Raw in negatively modulating DJNK signaling during closure (Byars, 1999).

To establish a regulatory link between raw and Jra, their epistatic relationship was determined. Embryos doubly mutant for raw and Jra were scored for the appearance of alternative dpp expression phenotypes (either missing from leading edge epidermal cells, as in Jra mutants or ectopic epidermal expression, as in raw mutants). The finding that dpp is not expressed in leading edge epidermal cells in raw;Jra double mutants defines Jra as epistatic to raw and confirms the hypothesis that raw functions upstream of the DJNK signaling pathway (Byars, 1999).

In summary, distinct features of functionality define raw as unique. The raw gene is the first of the dorsal-open group for which defects in gene expression have been documented in the amnioserosa. More notably, raw represents the first of the dorsal-open group mutants to show gross defects in dorsal closure that are attributable to a gain in DJNK signaling rather than a loss of DJNK signaling. The characterization of raw as a novel upstream component of the dorsal closure pathway represents an important first step in understanding the mechanism of regulating DJNK signaling during closure (Byars, 1999).

ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton

rib and raw mutations prevent cells in a number of tissues from assuming specialized shapes, resulting in abnormal tubular epithelia and failure of morphogenetic movements such as dorsal closure. Mutations of zipper, which encodes the nonmuscle myosin heavy chain, suppress the phenotypes of rib and raw, suggesting that rib and raw are not directly required for myosin function. Abnormal formation of the actin cytoskeletal structures underlying embryonic cuticular hairs suggests possible roles for rib and raw in organizing the actin cytoskeleton. The actin prehair structures are absent in rib mutants and abnormally shaped in raw mutants, indicating that the two genes have different functions required for organizing the actin cytoskeleton (Blake, 1999).

The fact that zip mutations suppress many of the mutant phenotypes of rib and raw is inconsistent with the hypothesis that either rib or raw is directly required for contraction of the actin cytoskeleton by myosin. Nevertheless, the suppression of the rib and raw phenotypes by zip mutations might be observed if the rib and raw products regulate myosin contraction either by repression or by controlling the direction of contraction. Alternatively, both the effect of the mutations on cell shape and the suppression by zip mutations could be observed if rib and raw contribute to remodeling of the actin cytoskeleton by involvement in the organization of the actin filaments. The counteraction of rib and raw mutant phenotypes by zip mutations could then occur if the normal activities of the rib and raw products on the cytoskeleton oppose to some extent the activity of myosin (Blake, 1999).

The effect of the rib and raw mutations on hairs and denticles of the embryonic cuticle offers support for the hypothesis that the gene products are active in organizing actin. In late embryogenesis, bundles of filamentous actin form epidermal extensions around which cuticular structures are secreted. Some of these cuticular structures are the external apparatus of sensory organs and others are nonsensory projections, the dorsal and lateral cuticular hairs and ventral denticles. The denticles and hairs, both sensory and nonsensory, have various shapes and orientations and are organized in stereotypical, segmentally repeated patterns. The actin cytoskeletal supports of the cell extensions can be observed in stage 16 and 17 embryos by staining with rhodamine labeled phalloidin. Both rib and raw mutations alter the morphology of the F-actin structures, but mutations of each gene have different effects on the structures (Blake, 1999).

In normal embryos actin bundles form a prehair in the cells that secrete sensory hairs, but no prehairs form in rib embryos. rib mutants lack hairs and denticles almost completely, leaving only a few isolated cuticular hairs and denticles. The remaining hairs are either much longer than normal or are abnormally curved. At junctions of three or more cells, rib embryos display intense actin spots, some of which could be sockets of sense organs. In addition, F-actin, which in wild-type epidermal cells accumulates in the cytoplasm and subsequently dissipates, remains in the cytoplasm of rib epidermal cells. The observation of cytoplasmic F-actin accumulation that disappears prior to formation of the actin prehair is consistent with the possibility that actin filaments begin to form in the cytoplasm and are recruited into the prehair structures. The rib product is apparently required for the formation of the larger actin structures from smaller actin filaments that form in the cytoplasm (Blake, 1999).

The cells of raw mutant embryos do form projections, albeit abnormally shaped ones. In raw mutants, hairs are generally disorganized in appearance, may be inappropriately clustered, and are often forked or branched. These are the same types of abnormalities described for embryos mutant for forked (f) and singed (sn), two genes that encode actin bundling proteins. Thus, the raw product could have a role in bundling or otherwise organizing actin filaments. The formation of the F-actin prehair structures might be independent of the activity of myosin. Zygotic expression of zip is not obviously required for formation of actin prehairs and predenticles since both form normally in zip mutants. If myosin does not affect the organization of actin into prehair structures, zip mutations would not be expected to alter the phenotype of rib mutants with respect to the failure of formation of prehairs. However, zip mutation suppresses the rib phenotype, causing a substantial increase in the number of denticles and hairs present on the embryonic cuticle of rib;zip mutants, as compared to rib mutants. Thus, zip counteracts the effect of rib mutations for each of the rib phenotypes. The suppression of the cuticular hair phenotype of raw mutations by zip is less obvious, but the severity of the branching and forking, characteristic of the raw prehair phenotype, is reduced in raw;zip double mutants. The fact that a zip mutation causes actin prehairs and predenticles to form more normally in rib and raw mutants indicates that myosin antagonizes the formation of the actin structures (Blake, 1999).

Although rib and raw have similar effects on the ability of cells to elongate, the differences in the effects of mutations of the two genes on the actin structures that underly cuticular hairs suggest that the two gene products have different functions with respect to the actin cytoskeleton. Distinct functions for rib and raw products are consistent with the observation that raw;rib double mutants are far more defective than embryos mutant for either of the genes individually. In the double mutants many of the affected tissues are greatly reduced in size and the embryos are generally very delicate. The extreme phenotype of the double mutant could be the result of separate defects in the actin cytoskeleton. The evidence presented provides further support for the hypothesis that rib and raw products have functions necessary for cytoskeletal activity, either in a structural or regulatory capacity. The data also indicate that the gene products are not required for myosin to apply force to the actin cytoskeleton. Because the products are essential for formation of the actin models of cuticular hairs and denticles, they could function directly in organizing actin filaments. Defects in reorganization of actin filaments of the cortical cytoskeleton could also explain the abnormal cell shapes associated with rib and raw mutants. However, as is the case with other rib and raw phenotypes, lack of zygotic zip activity suppresses the effect of mutations of the genes on hair and denticle formation in the embryonic cuticle. Therefore the rib and raw products could also act by repressing myosin or controlling its activity in some other way. Analysis of the rib and raw products will likely be necessary to resolve the issue (Blake, 1999).

The products of ribbon and raw are necessary for proper cell shape and cellular localization of nonmuscle myosin in Drosophila

Mutations in the genes rib and raw cause defects in the morphology of a number of tissues in homozygous mutant embryos. A variety of tubular epithelial tissues adopt a wide, round shape in mutants and dorsal closure fails. Cells of the normal tubular epithelia are columnar and wedge-shaped, and cells of the epidermis become elongated dorsoventrally as dorsal closure occurs. However, the cells of mutants are round or cuboidal in all of the tissues with mutant phenotypes, consistent with the hypothesis that the products of these genes are required for proper cell shape. Cytoskeletal defects, in particular, defects in myosin-driven contraction of the cortical actin cytoskeleton, could be responsible for the lack of specific cell shapes in mutant embryos. This possibility is supported by the observation that the intracellular localization of nonmuscle myosin to the leading edge of the dorsally closing epidermis is absent or reduced in rib and raw mutant embryos. In contrast, the band of actin that is also located at the leading edge is neither eliminated nor interrupted by either rib or raw mutations. Furthermore, mutations of zipper, the gene encoding the nonmuscle myosin heavy chain, exhibit mutant phenotypes in most of the same tissues affected by rib and raw, and many of the phenotypes are similar to those of rib and raw. Therefore, the products of rib and raw may be required for proper myosin-driven contraction of the actin cytoskeleton (Blake, 1998).

The genes raw and ribbon are required for proper shape of tubular epithelial tissues in Drosophila

The products of two genes, raw and ribbon, are required for the proper morphogenesis of a variety of tissues. Malpighian tubules mutant for raw or rib are wider and shorter than normal tubules, which are only two cells in circumference when they are fully formed. The mutations alter the shape of the tubules beginning early in their formation and block cell rearrangement late in development, which normally lengthens and narrows the tubes. Mutations of both genes affect a number of other tissues as well. Both genes are required for dorsal closure and retraction of the CNS during embryonic development. In addition, rib mutations block head involution, and broaden and shorten other tubular epithelia (salivary glands, tracheae, and hindgut) in much the same manner as they alter the shape of the Malpighian tubules. In tissues in which the shape of cells can be observed readily, rib mutations alter cell shape, which probably causes the change in shape of the organs that are affected. In double mutants raw enhances the phenotypes of all the tissues that are affected by rib but unaffected by raw alone, indicating that raw is also active in these tissues (Jack, 1997).


REFERENCES

Search PubMed for articles about Drosophila Raw

Bates, K. L., Higley, M. and Letsou, A. (2008). Raw mediates antagonism of AP-1 activity in Drosophila. Genetics 178(4): 1989-2002. PubMed ID: 18430930

Blake, K. J., Myette, G. and Jack, J. (1998). The products of ribbon and raw are necessary for proper cell shape and cellular localization of nonmuscle myosin in Drosophila. Dev. Biol. 203: 177-188. PubMed ID: 9806782

Blake, K. J., Myette, G. and Jack, J. (1999). ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton. Dev. Genes Evol. 209: 555-559. PubMed ID: 10502112

Byars, C. L., Bates, K. L. and Letsou, A. (1999). The dorsal-open group gene raw is required for restricted DJNK signaling during closure. Development 126(21): 4913-23. PubMed ID: 10518507

Jack, J. and Myette, G. (1997). The genes raw and ribbon are required for proper shape of tubular epithelial tissues in Drosophila. Genetics 147: 243-253. PubMed ID: 9286684

Jemc, J. C., Milutinovich, A. B., Weyers, J. J., Takeda, Y. and Van Doren, M. (2012). raw Functions through JNK signaling and cadherin-based adhesion to regulate Drosophila gonad morphogenesis. Dev Biol 367: 114-125. PubMed ID: 22575490

Lee, J., Peng, Y., Lin, W. Y. and Parrish, J. Z. (2015). Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw. Development 142: 162-173. PubMed ID: 25480915


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