ribbon: Biological Overview | Developmental Biology | Effects of Mutation | References
Gene name - ribbon
Cytological map position - 56C6--9
Function - transcription factor
Symbol - rib
FlyBase ID: FBgn0003254
Genetic map position - 2-88
Classification - BTB/POZ domain
Cellular location - nuclear
|Recent literature||Loganathan, R., Lee, J. S., Wells, M. B., Grevengoed, E., Slattery, M. and Andrew, D. J. (2015). Ribbon regulates morphogenesis of the Drosophila embryonic salivary gland through transcriptional activation and repression. Dev Biol. PubMed ID: 26477561
Ribbon (Rib) controls cell shape/volume increases during elongation of the Drosophila salivary gland (SG), without effects on general SG cell attributes such as specification, proliferation and apoptosis and without compromising epithelial-specific morphological attributes. To identify the genes regulated by Rib, ChIP-seq analysis was performed in embryos driving expression of GFP-tagged Rib specifically in the SGs. Microarray analysis compared RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, 60 genomic sites bound by Rib were identified that were likely to regulate SG-specific gene expression. Several of the identified Rib targets were identified by qRT-pCR and/or in situ hybridization. The results indicate that Rib regulates cell growth and tissue shape via a diverse array of targets through both transcriptional activation and repression. Furthermore, the results suggest that autoregulation of rib expression may be a key component of the SG morphogenetic gene network.
|Silva, D., Olsen, K. W., Bednarz, M. N., Droste, A., Lenkeit, C. P., Chaharbakhshi, E., Temple-Wood, E. R. and Jemc, J. C. (2016). Regulation of gonad morphogenesis in Drosophila melanogaster by BTB family transcription factors. PLoS One 11(11): e0167283. PubMed ID: 27898696
During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and SGPs to form a gonad with the proper architecture not only affects germ cell development, but can also lead to infertility. Therefore, it is critical to identify the molecular mechanisms that function within both the PGCs and SGPs to promote gonad morphogenesis. This study has characterized the phenotypes of two genes, longitudinals lacking (lola) and ribbon (rib), that are required for the coalescence and compaction of the embryonic gonad in Drosophila melanogaster. rib and lola are expressed in the SGPs of the developing gonad, and genetic interaction analysis suggests these proteins cooperate to regulate gonad development. Both genes encode proteins with DNA binding motifs and a conserved protein-protein interaction domain, known as the Broad complex, Tramtrack, Bric-a-brac (BTB) domain. Through molecular modeling and yeast-two hybrid studies, it was demonstrated that Rib and Lola homo- and heterodimerize via their BTB domains. In addition, analysis of the colocalization of Rib and Lola with marks of transcriptional activation and repression on polytene chromosomes reveals that Rib and Lola colocalize with both repressive and activating marks and with each other. While previous studies have identified Rib and Lola targets in other tissues, Rib and Lola are likely to function via different downstream targets in the gonad. These results suggest that Rib and Lola act as dual-function transcription factors to cooperatively regulate embryonic gonad morphogenesis.
During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. Mutations in the ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia. Directed cell migration of the salivary gland and dorsal epidermis are also affected in ribbon mutants. It is proposed that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements (Shim, 2001; Bradley, 2001).
In the wild type migrating tracheal branch, the basal surface of the tracheal epithelium is broad and smooth with an occasional pseudopodium extending from cells at the growing tip, much like those seen at the leading edge of migrating fibroblasts. As the basal surface extends toward the Bnl FGF signaling centers, the cell bodies and apical surface follow, and basal cytoplasmic extensions are never very prominent. In rib mutants, pseudopodia still extend from the basal surface, and are more numerous and pronounced than in wild type, occasionally forming extremely long processes that appear to reach their normal targets. This dissociation of the migration of the apical and basal tracheal surfaces is evident at stage 12 and continues for several hours into stage 14. At this later stage, the apical surface begins to deform in the direction of the basal cytoplasmic extensions, but even at this later stage the net distance traveled by the apical side is far less than in wild type. The apical defect is unlikely to reflect a general defect in apical-basal polarity of the tracheal epithelium because the apical determinant Crumbs, the apical marker TL1, and an apically localized mRNA are expressed and localize properly at the apical tracheal surface in rib mutants. It is concluded that rib mutations selectively affect movement and morphogenesis of the tracheal cell bodies and apical surface (Shim, 2001).
In ribbon mutant tracheae, the dorsal trunk fails to form, and ventral branches are stunted; however, directed migrations of the dorsal and visceral branches are largely unaffected. The dorsal trunk also fails to form when FGF or Wingless/WNT signaling is lost, and ribbon functions downstream of, or parallel to, these pathways to promote anterior-posterior migration. Directed cell migration of the salivary gland and dorsal epidermis are also affected in ribbon mutants, suggesting that conserved mechanisms may be employed to orient cell migrations in multiple tissues during development (Bradley, 2001).
Analysis of Wg signaling in tracheal branching suggests that cells are allocated to branches (cell allocation) independently from cell fate specification. (1) In Wingless signaling mutants the 'pre-dorsal trunk' cells are positioned correctly, but fail to migrate away from the transverse connective. (2) Wg signaling mutants do not express spalt, a dorsal trunk-specific marker. Thus, the cells are allocated to the dorsal trunk (DT), but do not express DT markers or behave like dorsal trunk cells. rib mutants, like Wg signaling mutants, also fail to form the DT, and 'pre-DT' cells are stalled at the transverse connective; however, unlike embryos lacking Wg signaling, rib mutants express sal in DT cells. Thus, rib is not required for cell allocation or cell fate specification (as monitored by sal), but is only required for branch migration. In summary, these observations suggest that, at least for the tracheal DT, cell allocation is independent of cell fate specification, and cell fate can be further subdivided into branch identity (controlled by genes such as sal that specify branch features) and branch migration, which involves rib (Bradley, 2001).
The similarity of the tracheal DT phenotypes in rib mutants and Wg signaling mutants raises the possibility that rib functions with Wg signaling for migration of DT cells. sal is the only known early downstream target of Wg signaling in the DT. Because the DT phenotype is more severe in embryos lacking Wg signaling than in sal mutants, there must be additional downstream targets of Wg signaling. Indeed, it can be predicted that these other genes control migration based on two findings. (1) DT cells are capable of migrating in sal mutants, but move in the wrong direction (dorsally). (2) When both Wg and Decapentaplegic signaling are activated in wild-type embryos (activated armadillo and activated thick veins in all tracheal cells), a complete longitudinal DT forms that does not express sal, suggests that sal may be dispensable for anteroposterior migration in some cases. Loss of rib results in a DT phenotype identical to that observed in loss of Wg signaling and rib functions in parallel to Wg-dependent sal expression. Together these results suggest that rib is working with Wg signaling, either in parallel or potentially as a downstream target, to direct DT migration (Bradley, 2001).
It is hypothesized that rib may respond to signals from multiple pathways based on analysis of a ventral cuticle phenotype. In rib mutants, the defects in ventral cuticle patterning appear most similar to the phenotype reported for the combined loss of late Wg signaling and Egfr signaling. In this tissue, rib could be integrating signaling from Wg and Egfr. In several other tissues requiring rib function, Wg signaling and signaling through a MAPK cascade are also required; however, in these cases, loss of either of the individual pathways results in phenotypes similar to those of rib mutants. For instance, rib is required for the cell shape changes in the leading edge cells during dorsal closure, a process that requires both Wg signaling and Jnk signaling. The second midgut constriction and the morphogenesis of the Malpighian tubules are defective in rib mutants, and both events also require both Wg and Egfr signaling. Similarly, in the trachea, rib could respond to Wg signaling and either of the two pathways (FGF or Egfr) that activate the MAPK cascade in tracheal cells. Since the rib phenotype is distinct from Egfr signaling mutants, a role for rib downstream of FGF signaling is favored. Indeed, the stalled outgrowth of all tracheal branches and stunted ventral branches observed in rib mutants may be linked to FGF signaling. Consistent with the idea that rib responds to MAPK signaling, the Rib protein has seven consensus MAPK phosphorylation sites (Bradley, 2001).
rib identifies a new step in primary branch budding and outgrowth that lies downstream or parallel to bnl signaling. What is this step? During primary branch budding, the basal surface of the tracheal epithelium is exposed to secreted Bnl/FGF, and the cells respond by extending pioneer cytoplasmic processes towards the Bnl source. But for a new branch to form, the cell bodies and apical surface must follow. Unlike isolated cells like Dictyostelium or fibroblasts migrating up a chemoattractant gradient, where the entire cell is exposed to the attractant, the cell bodies and apical surface of the tracheal epithelium are sealed off and do not have direct access to Bnl. These parts of the cell must receive the stimulus to move indirectly. rib might be required for transmission of the Bnl signal from the basal surface to the rest of the cell or to otherwise couple their movement. Alternatively, rib may be needed to propel the cell body forward once it receives the signal to move. Fibroblasts use distinct molecular mechanisms to move their leading and trailing sides forward; perhaps rib is required for a myosin-dependent process, like the one used to propel the trailing side of a migrating fibroblast forward. Rib might also be needed to promote the cell shape and apical surface changes necessary to form or extend a new tube. Although the data do not pinpoint the precise cellular event mediated by Rib, it is clear that the gene identifies a distinct step in tracheal branching one that may be common to a number of other epithelial morphogenetic events (Shim, 2001).
The genetic analysis indicates that rib functions downstream or parallel to the Bnl FGF pathway. If Rib functions downstream in the FGF pathway, for example, if its transcriptional regulatory activity is regulated by RAS/MAPK signaling like the Yan, DSRF and possibly Pointed transcription complexes, then this would provide a natural way of coupling morphogenetic events in the tracheal cell bodies and apical surface to events at the basal surface, where the Bnl signal is received. When the Bnl pathway is active, the receptor would directly stimulate outgrowth of the basal tracheal surface. But it would also indirectly stimulate migration and morphogenesis of the cell body and apical surface, via activation of Rib and induction of its target genes (Shim, 2001).
Rib, together with the Drosophila proteins Pipsqueak (Psq) and Tyrosine kinase related (Tkr), defines a new subfamily of BTB proteins containing Psq DNA-binding motifs. Although most other BTB domain proteins are also believed to function as transcription factors, this much larger subfamily of BTB domain proteins contains zinc-finger (ZF) DNA-binding motifs. The BTB domains of BTB/Psq proteins are no more similar to each other than they are to those of BTB/ZF proteins, although they are more similar to each other than they are to the BTB domain of Kelch, a cytoplasmic protein. The reason for the common coupling of BTB domains with two structurally unrelated DNA binding motifs, but not other DNA binding motifs, is unclear (Shim, 2001).
ribbon is thought to be required for generating specialized cell shapes. For instance, during dorsal closure, leading edge cells of the lateral epidermis fail to elongate in rib mutants. rib mutants also show abnormal dilation of salivary gland lumina in late embryogenesis, suggesting that either rib is also required at late stages to maintain organ shape or loss of early rib function indirectly causes the late lumenal dilation. rib appears to control cell shapes by regulating the cytoskeleton. During dorsal closure, a band of actin and myosin forms at the dorsal margin of leading edge cells. In rib embryos, the actin band is narrower and myosin heavy chain (MHC) is absent from leading edge cells. Thus, rib may be required for the localization or organization of cytoskeletal components. zip encodes a nonmuscle MHC and is required in many of the same tissues as rib; however, strong loss-of-function mutations in zip suppress the distended lumenal phenotype of rib salivary glands, suggesting that rib does not positively regulate myosin activities. Instead, rib may repress myosin contraction or regulate the direction of contraction, perhaps by providing a balancing force to the direction of basal myosin contractions. These studies reveal a role for rib in coordinating directed cell migration, a process that clearly involves actin/myosin dynamics. Thus, rib may modulate actin/myosin behavior for cell movement and cell shape during both tissue formation and tissue homeostasis. If rib is responding to signaling pathways, rib could be a critical factor linking signaling events to changes in the cytoskeleton (Bradley, 2001).
In addition to the tracheal system, rib is expressed in a number of other developing tissues, in a complex and dynamic pattern that coincides with various morphogenetic movements. Several sites of expression coincide with epithelial invaginations or evaginations to form tubes or sacs, as occurs during tracheal branch budding. These sites include the anterior and posterior midgut invaginations , salivary gland primordia as they invaginate and extend, stomodeum and proctodeum as they invaginate to form the foregut and hindgut, and Malpighian (renal) tubules as they bud. rib is also expressed during spreading of a planar epithelium, the epidermis, during dorsal closure. rib is expressed during or just after a number of mesenchymal-epithelial transitions, including the midgut mesenchyme as it reorganizes into an epithelium and forms the central portion of the gut (Shim, 2001).
Several of these morphogenetic events are defective in rib mutants. Indeed, rib mutants were first identified by their dorsal closure defect (Nusslein-Volhard, 1984). rib mutants also have defects in a number of other tissues including the Malpighian tubules, salivary glands and hindgut (Jack, 1997; Blake, 1998; Blake, 1999). Each of these are epithelia, and there are defects in the cell shape changes that underlie their morphogenesis. For example, rib mutant epidermal cells fail to elongate properly, and salivary gland cells fail to constrict apically (Shim, 2001).
Almost all epithelia undergoing morphogenetic movements face similar challenges as the tracheal epithelium during primary branching -- different parts of the epithelium must move coordinately although they are exposed to different environments. Based on the analysis of the tracheal function of rib, as well as its expression and activity in a variety of other morphogenetic processes, it is proposed that Rib is a key regulator of the movement and morphogenesis of epithelia, particularly events in the cell bodies or apical surface during budding of tubular epithelia. In some migrating epithelia, such as the spreading epidermis during dorsal closure, it is not clear that rib mutations specifically disrupt an apical process, so Rib may also influence epithelial migration and morphogenesis in other ways. Insight into the mechanisms and molecules that execute these morphogenetic events may come through the identification of transcriptional targets of Rib (Shim, 2001).
rib expression during development was analyzed by in situ hybridization of whole-mount embryos. rib transcript is detected in the developing tracheal system, as the tracheal precursor cells invaginate to form tracheal sacs and primary branches bud and grow out from the sacs. rib is also expressed in a variety of other tissues, in a complex and dynamic pattern that coincides with various morphogenetic movements. For example, rib is first detected at stages 5-8 (~2.5-3.5 hours AEL) in the primordia of the anterior and posterior midgut as the primordia invaginate to form tubes; expression is turned on again in the tubes a couple hours later (stages 10-14, ~5-11 hours AEL) as the tubes extend and form the central portion of the gut. rib expression is also seen in the developing mesoderm at or just after its ectodermal invagination and epithelial-to-mesenchymal transition, as the mesodermal cells begin their dorsal migration. rib is also expressed in the stomodeum and proctodeum, as these tissues invaginate to form the foregut and hindgut, and as the Malpighian tubules bud. rib is expressed broadly in the epidermis from stage 10 to 15 (~5-13 hours AEL) as it migrates during dorsal closure. Some of these sites of rib expression are also associated with morphological defects in rib mutants (Shim, 2001).
Although rib is expressed during many morphogenetic movements, some morphogenetic events are not associated with rib expression. For example, rib is not expressed strongly during cephalic furrow formation or formation of the anterior and posterior transverse folds (Shim, 2001).
Generation and maintenance of proper lumen size is important for tubular organ function. This study reports on a novel role for the Drosophila Rho1 GTPase in control of salivary gland lumen size through regulation of cell rearrangement, apical domain elongation and cell shape change. Rho1 controls cell rearrangement and apical domain elongation by promoting actin polymerization and regulating F-actin distribution at the apical and basolateral membranes through Rho kinase. Loss of Rho1 results in reduction of F-actin at the basolateral membrane and enrichment of apical F-actin, the latter accompanied by enrichment of apical phosphorylated Moesin. Reducing cofilin levels in Rho1 mutant salivary gland cells restores proper distribution of F-actin and phosphorylated Moesin and rescues the cell rearrangement and apical domain elongation defects of Rho1 mutant glands. In support of a role for Rho1-dependent actin polymerization in regulation of gland lumen size, loss of profilin (Chickadee) phenocopies the Rho1 lumen size defects to a large extent. Ribbon, a BTB domain-containing transcription factor, functions with Rho1 in limiting apical phosphorylated Moesin for apical domain elongation. These studies reveal a novel mechanism for controlling salivary gland lumen size, namely through Rho1-dependent actin polymerization and distribution and downregulation of apical phosphorylated Moesin (Xu, 2011).
Rho1 acts both in salivary gland cells and in the surrounding mesoderm to maintain apical polarity during gland invagination and to mediate cell shape change during gland migration. This study demonstrates a novel role for Rho1 in controlling salivary gland lumen size through regulation of actin polymerization and distribution and regulation of Moesin activity. By analyzing Rho1 alleles for which salivary gland cells invaginated and formed a gland, it was shown that zygotic loss of function of Rho1 results in shortening and widening of the gland lumen, which is accompanied by defects in cell shape change and cell rearrangement and failure of apical domains to elongate along the Pr-Di axis of the gland. These effects of Rho1 are mediated through Rok; inhibition of Rok completely phenocopies loss of Rho1 in these cellular events. Based on these studies, a model is proposed for Rho1 control of salivary gland lumen size, in particular lumen width, which is determined by cell rearrangement and apical domain elongation. Rho1 and Rok, through inhibition of cofilin, regulate cell rearrangement and apical domain elongation by promoting actin polymerization to localize F-actin at the basolateral membrane and by limiting the apical accumulation of F-actin. In parallel to its role in actin polymerization and distribution, Rho1 acts independently of Rok to limit apical p-Moe with Rib by an unknown mechanism and this function of Rho1 is specific for apical domain elongation. The data on cofilin (Twinstar) are consistent with those in cultured HeLa cells that showed that mammalian ROCK can inhibit cofilin activity indirectly through LIMK-mediated phosphorylation of cofilin (Xu, 2011).
Although manipulating Moe activity through gland-specific expression of MoeT559D was sufficient to completely phenocopy the Rho1 lumen defects, including cell rearrangement, it did so without disrupting actin polymerization or distribution. This is likely to be due to activated Moe strengthening the link between the actin cytoskeleton and the apical plasma membrane (without affecting levels of apical F-actin), which would increase apical membrane stiffness and remove the ability of gland cells to rearrange. Indeed, Moesin has been shown to control cortical rigidity during mitosis of cultured Drosophila S2R+ cells. Thus, Rho1 regulates cell rearrangement and apical domain elongation by controlling the actin cytoskeleton and Moesin activity through distinct mechanisms (Xu, 2011).
The observation that chic mutant glands phenocopy Rho1 mutant glands to a large extent, suggests that Rho1 control of salivary gland lumen size is mainly dependent on a requirement for Rho1 in actin polymerization. However, as the chic and Rho1 gland lumen phenotypes are not identical, with chic mutant glands lacking the apical accumulation of F-actin and p-Moe observed in Rho1 mutant glands, Rho1 probably has an additional function in limiting accumulation of F-actin and p-Moe at the apical membrane. This function of Rho1, at least for limiting apical F-actin, might partly involve Rab5- or Shi-mediated endosome trafficking, because inhibition of Rab5 alone or Shi alone led to accumulation of F-actin at the apical membrane. Although Rab5DN- or ShiDN-expressing salivary gland cells were enriched with apical F-actin, lumen size was not affected. This could be due to Rab5DN and ShiDN affecting a pool of apical F-actin distinct from that affected by Rho1 and/or because Rab5DN-expressing gland cells retain basolateral F-actin and the ratio of apical to basolateral F-actin is not altered sufficiently to cause lumen size defects. In Rho11B mutant gland cells, some early endosomes were not coated with F-actin. Actin is known to contribute to multiple steps of the endocytic pathway, including movement of endocytic vesicles through the cytoplasm and their transport to late endosomes and lysosomes. One possible mechanism by which Rho1 normally limits apical accumulation of F-actin is by promoting its removal from the apical membrane and accumulation on endocytic vesicles (Xu, 2011).
Currently, it is not know how Rho1 limits accumulation of apical p-Moe. Membrane localization and activity of Moesin can be regulated via a number of mechanisms, such as its phosphorylation on a conserved Threonine residue, binding to phosphatidylinositol-(4,5)bisphosphate [PtdIns(4,5)P2] and association with components of the sub-membrane cytoskeleton, such as Crb. Studies in cultured mammalian cells have demonstrated that Rho signaling activates Moe either through phosphorylation of Moe by ROCK or through ROCK-mediated inhibition of myosin phosphatase, which is known to dephosphorylate p-Moe. Although it is possible that Drosophila Rho1 positively regulates Moe activity by one or more of these mechanisms, this study shows that in the developing salivary glands Rho1 in fact negatively regulates Moe activity. In rib mutant embryos, in which p-Moe is enriched apically, salivary gland and tracheal cells showed decreased staining for Rab11 GTPase, which localizes to the apical recycling endosomes and to secretory vesicles destined for the apical membrane. Thus, Rho1, like Rib might limit apical p-Moe through its membrane transport (Xu, 2011).
In Drosophila imaginal disc epithelia, Moe negatively regulates Rho1 activity to maintain epithelial integrity and to promote cell survival. These studies demonstrating that in the developing salivary gland Rho1 antagonizes Moe activity by limiting its localization at the apical membrane, shed novel insight into the functional relationship between Rho1 and Moe. It is possible that in a dynamic epithelium, such as the developing salivary gland, Rho1 contributes to the precise spatial and temporal regulation of Moe activity to fine-tune selective changes in apical domain shape. By contrast, in the imaginal disc epithelium, Rho1 regulation of Moe might not be necessary and, instead, Moe regulation of Rho1 activity is required to maintain epithelial integrity and cell survival. Thus, Rho and Moe can antagonize each other's activities depending on the type of epithelia or cellular event (Xu, 2011).
Rescue studies with Rho1WT demonstrate that Rho1 functions predominantly in the salivary gland cells to control apical domain elongation and cell rearrangement. Interestingly, expression of Rho1WT in the mesoderm with twi-GAL4 has no effect on cell rearrangement and has little effect on apical domain elongation and lumen size, whereas it has been shown that Rho1WT expression in the mesoderm significantly rescues the gland migration defect of Rho11B mutant embryos. This suggests that gland migration and lumen size control are regulated by distinct mechanisms. In support of this conclusion, embryos mutant for multiple edematous wings, encoding the ?PS1 integrin subunit, which have defects in gland migration, show no defects in gland lumen width. Identifying the distinct and overlapping mechanisms by which salivary gland lumen width and length are controlled will help to elucidate the mechanisms by which lumen size is controlled in tubular organs (Xu, 2011).
In embryos doubly mutant for Wg and Dpp signaling, only visceral branch (VB) cells migrate. Similarly, in embryos doubly mutant for rib and thick veins (tkv), which encodes one of the receptors essential for Dpp signaling, only VB cells migrate, a phenotype identical to that of embryos doubly mutant for tkv and armadillo (arm), which encodes an essential component of Wg signaling. Thus, like Wg signaling, rib plays an instructive role in DT formation (Bradley, 2001).
In spalt (sal) mutants, dorsal trunk (DT) cells migrate dorsally instead of forming the DT, whereas in Wg signaling mutants, DT cells are stalled at the transverse connective (TC). Thus, WG signaling must regulate other genes, in addition to sal, that control migration. Given that rib appears to phenocopy the loss of WG signaling in the DT, and that rib functions downstream of or in parallel to Wg signaling, rib itself might be a target of Wg signaling. rib RNA is expressed throughout the epidermis and is not obviously upregulated in the trachea. Thus it is unlikely that rib is transcriptionally controlled by Wg or other signaling pathways (Bradley, 2001).
rib mutants fail to complete dorsal closure, the process by which the cells of the lateral epidermis move dorsally to encompass the amnioserosa and seal the dorsal surface of the embryo. The Jun N-terminal kinase (Jnk) signaling pathway and the Wg signaling pathway are required for dorsal closure. Both pathways are necessary for the characteristic cell shape changes in the leading edge cells and the transcriptional activation of dpp. rib mutants also lack the characteristic elongation of the cells at the leading edge, and at late stages, these cells are large and misshapen (Blake, 1998; Bradley, 2001).
To determine whether the dorsal closure defects in rib mutants are related to defects in Jnk or Wg signaling, the dorsal cuticle of larvae carrying different allelic combinations of rib mutations was examined. In the allelic combinations that could be scored (i.e., those in which sufficient cuticle was produced), approximately two-thirds of the larvae had a large dorsal hole, and one-third had a small anterior dorsal hole with a puckering of the remaining dorsal cuticle. This range of phenotypes is similar to the defects in larvae with loss-of-function mutations in either Jnk or Wg pathway components. Whether dpp expression is maintained in the leading edge cells of rib mutants was investigated. Unlike mutations in either Jnk or Wg signaling components, in which dpp expression is absent in leading edge cells, dpp expression is observed at high levels in the leading edge cells in rib mutants. At late stages, dpp expression is often observed in lateral patches. This apparent increase of dpp expression in rib mutants could be due to increased numbers of cells expressing dpp, increased size of leading edge cells and/or loss of cell cohesion (which could cause cells to collapse or remain in more ventral positions). In any case, this experiment reveals that, as in the trachea, rib is not an upstream activator of Jnk or Wg signaling and that Jnk- and Wg-dependent activation of dpp is not mediated by rib. Mutations in rib cause defects at an earlier step in dorsal closure than dpp mutations: the leading edge cells fail to change shape in rib mutants, whereas Dpp signaling is required for cell shape changes and movement of the ectodermal cells just ventral to those at the leading edge. Thus, if rib functions downstream of the Jnk or Wg pathway to mediate dorsal closure, it must be acting in parallel to dpp activation (Bradley, 2001).
Patterning in the ventral cuticle is also impaired in rib larvae, which exhibit both a narrowing of the lateral extent of denticle belts and a fusion of belts at the midline. At a gross level, these phenotypes are similar to those described for the Egfr signaling mutants, rho and spitz. The cuticle phenotypes of different allelic combinations of rib mutations were examined, scoring both the lateral extent of denticle belts and denticle diversity. The lateral extent of rib denticle belts is narrowed to 37%, 46% and 70%-100% of the wild-type width, consistent with an allelic series (rib2/rib2<rib2/rib1=rib2/Df<rib1/rib1=rib1/Df). rib1/rib1 and rib1/Df cuticles were often very hard to detect, suggesting that very little cuticle is secreted (Bradley, 2001).
The loss of denticle diversity in rib mutants also corresponds to the above allelic series. The least affected cuticles had the most diversity of denticle types (rib2/rib2), whereas more severely affected cuticles had only one or two denticle types (rib2/rib1 and rib2/Df), and the most severely affected cuticles had very few faint denticles that appeared to be of a single type (rib1/rib1 and rib1/Df). The denticle belts of rib larvae with a single denticle type looked notably similar to larvae simultaneously lacking the late activities of Wg and Egfr signaling, in which all denticles are type 5 (wgts, UAS-DN-DER, arm.Gal4). Unlike Wg/Egfr-deficient larvae, however, not all of the denticles in rib mutants are oriented posteriorly. Overall, the dorsal and ventral cuticle phenotypes, together with the tracheal defects, suggest that rib may function with a combination of signaling pathways. It is clear that rib does not function upstream of these pathways, nor does rib interfere with transcriptional activation of early target genes. Thus, rib functions downstream of or parallel to these pathways to promote cellular changes (Bradley, 2001).
Signaling pathways controlling cell migration in the embryonic salivary gland have not yet been identified. Nonetheless, the salivary gland, like the tracheal system, invaginates through a stereotypical process involving directed cell migration. The salivary glands form from two paired primordia that arise from the ventral ectoderm of parasegment two. Through changes in cell shape and migration, the primordia are internalized and ultimately give rise to two cell types: secretory and duct. The secretory cells are the first to invaginate and proceed in an ordered, sequential manner beginning with the cells in the dorsal posterior region of the primordium. The secretory cells move dorsally into the embryo, then turn and migrate posteriorly until the distal half of the gland reaches the level of the third thoracic segment. After the movements of head involution, the salivary glands lie closer to the anterior end of the embryo and are oriented along the anteroposterior axis. Concomitant with later secretory cell migrations, the duct cells undergo a complex set of morphogenetic movements to create a tubular structure. This tube starts at the larval mouth and then branches to connect to the two secretory glands (Bradley, 2001).
Although salivary glands have been reported to be abnormal in late stage rib mutant embryos, earlier stages were not analyzed. As with the tracheal primordia, the secretory gland primordia in rib mutants are indistinguishable from those in wild-type embryos, and the initial invagination proceeds normally; however, rib secretory cells do not migrate past the point at which wild-type cells turn and migrate posteriorly. Thus, the secretory cells in rib mutants never reach their final destination. At late stages, the lumina of the salivary glands are greatly enlarged, compared to wild-type glands, suggesting that rib may also play a role in maintaining organ shape once the salivary gland has formed. Expressing a rib transgene specifically in the secretory cells of rib mutants restores both directed migration and lumen size. Thus, rib function is required in secretory cells to control migration and organ shape (Bradley, 2001).
The salivary duct also fails to undergo proper morphogenesis in rib mutants. Two duct markers, Trh protein and BTL mRNA, are detected in a normal pattern in the duct primordia of rib mutants. In contrast, duct cells stain poorly for the Dead ringer (Dri) protein, which is normally expressed robustly by stage 13; only diffuse low levels of Dri expression are detected in rib mutants prior to stage 15. In late stage rib mutant embryos, no tubes or rudimentary individual tubes connected to the secretory glands were seen; these semi-tubular structures do not elongate and never elaborate into a normal duct. In embryos expressing a rib transgene in secretory cells of rib mutants, duct formation is restored. This result indicates that rib duct defects are indirect and suggests that duct formation requires proper secretory cell morphogenesis. While both salivary gland structures are abnormally formed in rib mutants, there is a specific requirement for rib in the secretory cells for their posterior migration, similar to the requirement for rib in the tracheal DT cells for their anteroposterior migration (Bradley, 2001).
In the more severe allele rib1, a single nucleotide change in the rib ORF results in a nonsense codon after residue 282. This mutation deletes the entire C-terminal half of the protein, and is likely to be null, consistent with phenotypic analysis. rib2 has a single base change that replaces arginine 58 with a histidine (R58H) in the BTB/POZ domain. A mutation in rib was also discovered on the zipper1 (zip1) chromosome (Blake, 1998). zip1 mutants fail to complement both rib alleles; the rib ORF on the zip1 chromosome was sequenced and the identical nucleotide change was found that created the R58H mutation in rib2. This result is consistent with the phenotypic report that, when recombined off the zip1 chromosome, the ribz1 allele behaves like rib2 and is not as severe as rib1 (Blake, 1998). It is not clear whether the zip1 chromosome recombined with rib2 at some point, or whether finding the identical residue substitution indicates the importance of R58 in RIB function. All other detected base changes that resulted in residue substitutions were detected on all rib chromosomes and/or on the balancer chromosome, suggesting that these other changes are polymorphisms not responsible for rib phenotypes (Bradley, 2001).
Imprecise excision of the Terminal-3 P[w+,lacZ] tracheal marker at cytological position 56A-B generates a mutation (ex12) that severely impairs tracheal branch outgrowth. In wild-type embryos, all primary branches have grown out and the dorsal trunk branches have fused by stage 13, as seen by staining with tracheal antiserum TL1. In ex12 mutants, there is little branch outgrowth, and the trachea remain as a series of elongate, mostly unbranched sacs, similar to bnl null mutants at this stage. The block in branch outgrowth in ex12 mutants is not absolute because after stage 14, although 99% of tracheal branches remain stalled, 1% form and grow out excessively and in aberrant directions. By stage 14, several other morphogenesis defects become apparent, including failure of epidermal dorsal closure and midgut constriction formation (Shim, 2001).
The TL1 tracheal antiserum stains the apical surface and lumen of the developing tracheal epithelium. To further characterize the tracheal migration defects, rib1, rib2, and ribex12 mutants were examined using the tracheal cytoplasmic marker 1-eve-1, a viable P[lacZ] insert in trachealess. Despite the severe defect in movement of the apical tracheal surface, the basal surface continues to extend actively in the mutants (Shim, 2001).
Whether Bnl FGF signaling is affected in rib mutants was examined. In situ hybridization for bnl and btl transcripts showed that both are expressed grossly normally in ribP7 embryos, a null allele of rib. Furthermore, unlike bnlP1 mutants, in which only an occasional tracheal cytoplasmic extension was detected, in rib mutants cytoplasmic extensions form and grow towards their normal targets, implying that the Bnl pathway is active (Shim, 2001).
To examine more directly whether rib tracheal cells can sense and respond to Bnl, downstream components in the Bnl signaling pathway were examined. Expression of the terminal branch gene blistered/DSRF, which is normally induced by Bnl signaling at the ends of growing primary branches, is not induced to high levels in rib mutants. However, this appears to be a secondary consequence of the migration defect, because when a bnl transgene (UASbnl) is expressed ubiquitously in rib mutants to expose the stalled tracheal cells to high concentrations of Bnl, MAPK is activated and blistered/DSRF expression is induced throughout the tracheal epithelium, as in rib+ animals. It is concluded that rib mutant tracheal cells can respond to Bnl. This suggests that the migration defect results from either an inability to transmit the Bnl signal from the basal surface to the cell bodies and apical surface, or an inability of the cell bodies and apical surface to respond once they receive the signal (Shim, 2001).
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).
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).
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).
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).
Search PubMed for articles about Drosophila ribbon
Bates, K. L., Higley, M. and Letsou, A. (2008). Raw mediates antagonism of AP-1 activity in Drosophila. Genetics 178(4): 1989-2002. PubMed Citation: 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(1): 177-88. 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 Citation: 10502112
Bradley, P. L. and Andrew, D. J. (2001). ribbon encodes a novel BTB/POZ protein required for directed cell migration in Drosophila melanogaster. Development 128(15): 3001-15. 11532922
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 Citation: 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. 9286684
Nusslein-Volhard, C., Wieschaus, E. and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster: I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193: 267-282
Shim, K., Blake, K. J., Jack, J. and Krasnow, M. A. (2001). The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis. Development 128(23): 4923-33. 11731471
Xu, N., Bagumian, G., Galiano, M. and Myat, M. M. (2011). Rho GTPase controls Drosophila salivary gland lumen size through regulation of the actin cytoskeleton and Moesin. Development 138(24): 5415-27. PubMed Citation: 22071107
date revised: 10 February 2010
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