scarface: Biological Overview | References
Gene name - scarface
Cytological map position - 41F6-41F8
Function - enzyme (inactive)
Symbol - scaf
FlyBase ID: FBgn0033033
Genetic map position - 2R:1,564,254..1,579,200 [-]
Classification - serine-type endopeptidase activity
Cellular location - secreted
In Drosophila, dorsal closure is a model of tissue morphogenesis leading to the dorsal migration and sealing of the embryonic ectoderm. The activation of the JNK signal transduction pathway, specifically in the leading edge cells, is essential to this process. In a genome-wide microarray screen, new JNK target genes during dorsal closure were identified. One of them is the gene scarface (scaf), which belongs to the large family of trypsin-like serine proteases. Some proteins of this family, like Scaf, bear an inactive catalytic site, representing a subgroup of serine protease homologues (SPH) whose functions are poorly understood. This study shows that scaf is a general transcriptional target of the JNK pathway coding for a secreted SPH. scaf loss-of-function induces defects in JNK-controlled morphogenetic events such as embryonic dorsal closure and adult male terminalia rotation. Live imaging of the latter process reveals that, like for dorsal closure, JNK directs the dorsal fusion of two epithelial layers in the pupal genital disc. Genetic data show that scaf loss-of-function mimics JNK over-activity. Moreover, scaf ectopic expression aggravates the effect of the JNK negative regulator puc on male genitalia rotation. scaf acts as an antagonist by negatively regulating JNK activity. Overall, these results identify the SPH-encoding gene scaf as a new transcriptional target of JNK signalling and reveal the first secreted regulator of the JNK pathway acting in a negative-feedback loop during epithelial morphogenesis (Rousset, 2010).
The trypsin- and chymotrypsin-like (S1 family) serine proteases (SPs) are mainly represented in animals and are implicated in food digestion, blood coagulation, immune response and development. They are almost exclusively localized outside the cytoplasm to perform their function and they are synthesized with a signal peptide for secretion. In addition, SPs are usually produced in an inactive form, which is cleaved upon a signal to form a functional protease. This mechanism favours a rapid and local response to a stimulus. For example, in mammals, cascades of localised SP activities control emergency blood clotting at a wound site. During Drosophila development, dorsoventral patterning of the embryo results from an extracellular cascade involving the SPs Nudel, Gastrulation defective, Snake and Easter and leading to the activation of the Toll pathway in the ventral region (Rousset, 2010).
SPs are endopeptidases that use a serine for their catalytic activity and are characterized by the presence of the residues histidine, aspartic acid and serine (HDS) in the active site. Serine protease homologues (SPHs) are also found in various animal genomes and are defined by the lack of at least one of the essential residues of the catalytic triad HDS. Only a few SPHs have been characterized. One well-known example in mammals is the Hepatocyte growth factor or Scatter factor (HGF, SF). In Drosophila, almost 30% of the 204 identified SP genes encode SPHs that are therefore most probably catalytically inactive. Only four of these SPH genes have been assigned a function. Mutations in masquerade (mas) lead to defects in somatic muscle attachment and in the formation of the nervous system during embryogenesis. The other three genes, spheroide, sphinx1 and sphinx2, were identified in an RNAi screen aimed at identifying genes involved in immunity (Rousset, 2010).
This study isolated the SPH encoding gene scarface (scaf) in a microarray screen designed to identify new genes transcriptionally regulated by the JNK (c-Jun N-terminal kinase) pathway during dorsal closure (DC) of the Drosophila embryo. DC is a major morphogenetic movement that takes place after germ-band retraction to close the eye-shaped opening of the dorsal embryonic epidermis, over the transient tissue called the amnioserosa (AS). Cell elongation is responsible for the dorsalward spreading of the lateral epithelial sheets. The AS, conversely, progressively reduces through cell constriction and engulfment accompanied by cell death. Precise activation of the JNK pathway, specifically in the dorsalmost row of epithelial cells in contact with the AS (the leading edge, LE), is a prerequisite for DC progression. Several transcriptional targets of the JNK pathway have already been described during DC, such as the profilin-coding gene chickadee, the transcription factor cabut, the integrin-coding genes scab and myospheroid or the trafficking gene Rab30. However, only two target genes are specifically expressed in the LE, decapentaplegic (dpp) and puckered (puc). Dpp, of the TGFβ family, participates in cell spreading of the lateral epithelium, cell constriction in the AS and connection of these two tissues. Dpp also regulates remodelling of the cytoskeleton in the segment border cells by activating Cdc42 and dPak. The gene puc encodes a phosphatase that sets up a negative-feedback loop at the level of the c-Jun kinase Basket (Bsk). In addition, other JNK target genes are involved in different biological processes. For instance, JNK-induced expression of matrix metalloproteinases is required for disc eversion and tumour invasion; JNK protection against oxidative stress is realized through expression of autophagy-related genes; and during immune response, JNK participates in the expression of antimicrobial peptide genes (Rousset, 2010).
This paper reports the characterization of scaf during Drosophila development. The catalytic triad of the SP domain of Scaf lacks two of the conserved HDS residues and Scaf was thus classified as a SPH. Although a previously isolated semi-lethal mutation led to adult escapers exhibiting a head scar under the proboscis (Bonin, 2004), no function has been attributed to scaf. This study shows that the JNK signalling pathway regulates scaf expression in the embryo and in imaginal discs. In S2 cells and in vivo, the protein Scaf is secreted, indicating that it behaves as an extracellular SPH. Loss-of-function and gain-of-function in vivo studies uncovered an essential role of scaf during epithelial morphogenesis through antagonistic regulation of JNK signalling (Rousset, 2010).
This study shows that scaf is specifically induced by the JNK pathway in the LE cells that drive embryonic DC. This is the third gene, after dpp and puc, showing this very specific site of expression under the control of JNK. In addition, scaf expression has been demonstrated to be regulated by JNK in the A8 segment of the male genital disc. Scaf is also detected in the JNK-responsive cells (expressing puc-lacZ) of wing and leg discs, indicating that scaf can be considered as a general JNK target gene during epithelial morphogenesis. Like other proteins of the SP family, Scaf is a secreted SPH. Importantly, the results point to a negative role of scaf against JNK activity during DC in the embryo and during male terminalia formation in the pupa. Therefore, in addition to the inducible antagonist puc, the JNK pathway restrains its own activity during epithelial morphogenesis through the expression of the SPH gene scaf (Rousset, 2010).
The amount of trypsin- and chymotrypsin-like SP enzymes in metazoan genomes is highly variable, from a dozen in C. elegans to more than two hundred in dipteran species like Drosophila and about a hundred in mammals. Gene expansion that occurred in flies is remarkable and concerns both the active enzymes and their homologues, indicating specific adaptations to environment and life strategies. Catalytic domains of SP enzymes share high structural similarities and substrate specificity is insured by a binding cleft located near the active site, as well as surface loops present in the SP domain. SPH proteins have lost their catalytic activities and it is assumed that they have adopted new functions based on novel binding capacities. For instance, SPH could compete with an active SP for specific substrates (Rousset, 2010).
Classical genetics has revealed the implication of various genes in DC and other studies have identified several JNK target genes. However, scaf is the first member of the SP family with a role in this epithelial sealing movement. Like for the Drosophila SPH gene mas, mutations in scaf did not show complete expressivity and penetrance in the embryo. One straightforward explanation is the presence of a redundant protein among the various other SPH of the Drosophila genome. Compensatory mechanisms, which are known to be important during DC, could also hide the impact of scaf removal. Nevertheless, the results showed that scaf is important for morphogenesis by directly modulating the activity of the JNK pathway. How scaf negatively acts is still an open question, but it is probable that Scaf plays a role in the extracellular space, in contrast to the strong intracellular inhibitor Puc. The signal that activates JNK signalling (and therefore DC) has not yet been discovered, preventing a straightforward analysis of Scaf molecular action. SP proteins are well-described for their role in activating signal transduction pathways or in degrading the extracellular matrix (ECM), and defects in muscle attachment observed in mas mutations might be due to a decrease in cell-matrix adhesion. Accordingly, possible roles of Scaf could be to act locally on an unknown extracellular signal or at the level of the putative receptor of the JNK pathway, through interaction with the ECM. Functioning in a negative-feedback loop, scaf would allow a fine tuning of signalling in JNK-active tissues (Rousset, 2010).
The JNK signalling pathway is a major player of epithelial morphogenesis in many organs and developmental stages and its activity must be precisely controlled to coordinate tissue sealing. Two JNK target genes, puc and scaf, code for antagonists and therefore participate in this control. Whereas Puc acts intracellularly, Scaf is likely to operate in the extracellular space and represents the first secreted regulator of JNK-dependent epithelial sealing. Further studies of the role of scaf will highlight how a signal transduction pathway synchronizes collective cell behaviour in a three-dimensional environment, and how these cell rearrangements are controlled extracellularly to lead to a precise coordination of the movement (Rousset, 2010).
Cell-matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. This study analysed the role of the secreted serine protease-like protein Scarface in germ-band retraction and dorsal closure -- morphogenetic processes that rely on the activity of integrins and laminins. Evidence that scarface is regulated by c-Jun amino-terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. It is proposed that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context (Sorrosal, 2010).
During germ band retraction (GBR), the tail end of the germ band, or embryo proper, interacts with the amnioserosa (AS), an epithelium of large flat cells that does not contribute to the larva, and moves to its final posterior position. After GBR, the ectoderm has a gap on its dorsal side that is occupied by the amnioserosa (AS). The dorsal-most cells of the ectoderm, on both sides of the embryo, are in contact with the AS and are called leading edge (LE) cells. During DC, LE cells direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS until they meet and fuse at the dorsal midline. Attention of this study centered on scarf on the basis of its embryonic expression pattern. Two piggyBac insertions, scarf PBss(GFP) and scarf M13.M2(lacZ), were expressed at a high level in LE cells during GBR and DC and at a low level in AS cells. The expression of scarf was confirmed by in situ hybridization. It was also expressed in the head and tail regions, and in the ventral ectoderm in a segmental pattern (Sorrosal, 2010).
The JNK pathway is activated in LE cells and leads to the expression of the signalling molecule Dpp and the phosphatase Puckered (Puc). The scarf gene was expressed in the same cells as puc and dpp at the LE. Reduced JNK - Basket (Bsk) in Drosophila - or expression of a dominant-negative version of Bsk or Puc, which mediates a feedback loop repressing JNK activity, led to the loss of scarf expression in LE cells. Loss of puc activity, which leads to increased levels of JNK activity, or expression of an activated version of JNK-activating kinase (Hemipterous in Drosophila), led to the expansion of the scarf expression domain throughout the lateral ectoderm. Dorsal cells had a stronger response to increased JNK, suggesting that JNK requires the activity of another factor expressed in this region to induce scarf. As stated, the JNK cascade drives the expression of the secreted molecule Dpp. In embryos mutant for the Dpp receptor Thickveins, scarf expression at the LE was not lost and ectopic activation of the Dpp pathway did not induce the ectopic expression of scarf. These results indicate that scarf expression is induced by JNK in LE cells (Sorrosal, 2010).
Homozygosis or trans-heterozygosis for the piggyBac insertions scarf PBss and scarf M13.M2 caused semi-lethality and survivors had scars on the head, phenotypes that were reverted by precise excision of the transposon elements. As embryos showed no obvious cuticle phenotype, stronger alleles of scarf were generated by imprecise excision of a P element located in the third intron of scarf (scarf KG05129). One allele (scarf Δ1.5) was isolated that led to the loss of scarf messenger RNA expression. This is an embryonic lethal allele and trans-heterozygous combinations of scarf Δ1.5 or Df(2R)nap14, a deficiency that uncovers the scarf locus, with scarf PBss or scarf M13.M2 were semi-lethal and resulted in scars on the head. Homozygous animals for scarf Δ1.5 or Df(2R)nap14 showed similar phenotypes. Their larval cuticles were either dorsally wrinkled or presented a dorsal hole or a U-shaped phenotype, which is suggestive of failures in GBR and DC. A high proportion of embryos showed undifferentiated cuticles. Ubiquitous expression of scarf largely rescued the scarf Δ1.5 phenotype (Sorrosal, 2010).
Time-lapse recordings indicated that these embryos were delayed in development and showed a failure in GBR. Frequently, the attachment of the AS to the tail end of the germ band was compromised and the germ band did not retract. In embryos that were able to retract, DC started and LE cells began to direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS. However, either the AS detached from the neighbouring epidermal sheet or the epidermal sheets met and fused at the dorsal midline but the dorsal epidermis reopened. The requirement of scarf in DC was analyzed in greater detail in fixed staged embryos. During the initial steps of DC, lateral ectodermal cells start to elongate dorsally and an actin cable at the LE is formed, which helps to generate a linear fence at the interface between LE and AS cells. In scarf Δ1.5 embryos, elongation of the lateral ectoderm was compromised frequently, the actin cable was not formed properly, the interface between LE and AS cells became irregular, and eventually rips appeared at the AS-LE interface. The elongation of lateral ectodermal cells and adhesion between AS and LE cells are mediated by the activity of Dpp and JNK activity controls the polymerization of actin into a cable at the LE. In embryos lacking scarf, the activity of the Dpp and JNK signalling pathways was not affected. Thus, Scarf has a role in DC without affecting the activity levels of these pathways (Sorrosal, 2010).
Integrins and laminins have a fundamental role in GBR and DC by mediating interactions between AS cells and neighbouring cell populations. As defects were observed in these processes on scarf depletion and a reduction in scarf levels increased the frequency of cuticle phenotypes caused by depletion of the β-integrin position-specific (βPS) subunit, the contribution of Scarf to the regulation of the expression levels and subcellular localization of these proteins was examined. Cross-sectional views of properly staged wild-type and scarf Δ1.5 embryos stained for βPS integrin revealed similar levels and localization of integrins. The JNK signalling regulates the expression of the βPS integrin subunit during DC. Although ectopic activation of JNK induced increased expression of βPS integrin, ectopic expression of scarf did not exert this effect. The expression of LanA, one of the two existing fly α-laminins. In wild-type embryos, high levels of LanA were localized at the BM of AS cells. Interestingly, scarf embryos revealed strong defects in the polarized distribution of LanA. It was localized on the apical side of the AS epithelium and its protein levels were largely reduced in the BM. Ubiquitous expression of scarf largely rescued the levels of LanA at the BM. Similar defects were observed in the polarized distribution of LanA in the lateral ectoderm of scarf mutant embryos, and these defects were also rescued largely by the ubiquitous expression of scarf. Apico-basal polarity of AS cells, visualized by the basal localization of βPS integrin and the localization of E-cadherin (E-cad) at the adherens junctions, was not affected in hypomorphic scarf PBss and scarf M13.M2 embryos and in some scarf Δ1.5 embryos even though LanA was localized on the apical side. However, scarf depletion was frequently found to cause defects in the apico-basal polarity of AS cells and this was accompanied by multilayering of the AS epithelium. These observations suggest that before inducing a loss of epithelial integrity, the absence of Scarf caused aberrant localization of LanA on the apical side of the AS epithelium and reduced LanA in the BM, without affecting the distribution of apical and baso-lateral proteins. As integrins are involved in maintaining epithelial polarity, these results suggest that the observed loss of cell polarity is a consequence of reduced integrin-mediated adhesion to the BM. Consistent with this view, defects in the attachment of the AS cells to the underlying yolk cell were observed frequently (Sorrosal, 2010).
Although the defects observed in scarf Δ1.5 embryos resemble those caused by βPS integrin depletion, only defects in the attachment of the AS with the underlying yolk cells are found in lanA mutant embryos. Mutations in wing blister (wb), the only other fly α-laminin, cause defects in GBR and in the attachment between AS cells and the posterior germ band. These data suggest that LanA and Wb have a redundant function and that Scarf most probably promotes BM localization of not only LanA but also Wb. Consistent with the proposed redundancy, halving the dose of lanA or wb increased the frequency of the βPS integrin loss-of-function cuticle phenotype. The expression of Wb protein in AS cells using the available antibodies and the role of Scarf in the polarized distribution of other BM proteins, such as Perlecan or Collagen IV, were not addressed as these two proteins were not expressed in AS cells (Sorrosal, 2010).
These results indicate that Scarf depletion causes defects in the BM localization of LanA and in epithelial apico-basal polarity. The defects resemble those observed in follicle cells (that is, epithelial cells covering the fly oocyte) mutant for crag, a gene encoding for a protein localized in early and recycling endosomes and proposed to regulate protein transport and membrane deposition of BM proteins. Zygotic removal of crag induced anomalous localization of LanA on the apical side of AS cells. Zygotic or both maternal and zygotic removal of crag produced cuticles with a weak dorsally wrinkled phenotype, suggesting that Crag has a redundant function with another protein in the fly embryo and that the low BM levels of LanA observed in these embryos are sufficient to exert their function. Interestingly, halving the dose of scarf expression gave rise to a large proportion of crag cuticles with phenotypes similar to those caused by scarf depletion (Sorrosal, 2010).
Antibodies against Scarf were raised to analyse its subcellular localization. As the antibodies did not work properly at embryonic stages and did not detect endogenous levels of Scarf protein, the wing imaginal disc, a monolayered epithelium, was used to analyse its subcellular localization. Scarf was not detected in wild-type wing cells. When scarf and green fluorescent protein (GFP) were expressed in a restricted domain in the wing disc, Scarf was detected not only in the GFP-labelled scarf-producing cells, but also on the apical side of the epithelium at long distances from the source. When atrial natriuretic factor-GFP (a fusion between the secreted rat atrial natriuretic peptide and GFP) was expressed, the protein was also observed at long distances from the source; however, it did not accumulate on the apical side of the epithelium. Scarf was found to localize in small punctate structures throughout the cytoplasm of scarf non-producing cells. These structures corresponded either to Rab5-positive early endosomes, to Rab7-positive late endosomes, or to Rab11-positive recycling endosomes. Together, these results indicate that Scarf is secreted apically and is internalized through endocytosis by non-producing cells. To confirm that Scarf is a secreted protein, Drosophila S2 cells were transfected with either a scarf-myc-tagged transgene or a transgene driving the expression of a myc-tagged membrane-tethered form of Scarf (Scarf-CD2). Scarf was isolated not only from the protein extract of the scarf-myc-expressing cells but also from the supernatant. Transfected Scarf-CD2 and endogenous actin were not observed in the supernatant (Sorrosal, 2010).
This study has characterized the role of scarf, a JNK-regulated gene, in GBR and DC, two morphogenetic processes that rely on cell-matrix interactions between the AS epithelium and neighbouring cell populations. Evidence that Scarf is a secreted protein expressed in LE cells and involved in promoting the localized accumulation of LanA in the BM of AS cells. Although low levels of Scarf were detected in AS cells, the cuticle phenotype of scarf mutant embryos and the defects observed in BM localization of LanA and epithelial integrity of AS cells were rescued largely by driving expression of a scarf transgene only in ectodermal cells, indicating that Scarf is acting as a secreted protein. Three alternative hypotheses might explain the role of Scarf and Crag in the polarized localization of LanA to the BM. As Crag and Scarf are localized specifically on the apical side of the epithelium, they would have a repulsive role, inhibiting the targeting of LanA-containing vesicles with apical membranes. Alternatively, LanA might be secreted apically and basally and be stabilized preferentially on the basal side or degraded on the apical side. As Scarf encodes for a putative serine protease-like protein without catalytic activity, Scarf would function, in this scenario, to facilitate the effect of a cascade of serine proteases involved in the degradation of LanA in the apical domain of epithelial cells. Finally, in scarf mutant cells, LanA might be transported from the basal to the apical side through transcytosis, a mechanism responsible for the formation of a small apical BM cap over pre-invasive epithelial cells during Drosophila oogenesis (Sorrosal, 2010).
Another secreted serine protease-like protein without catalytic activity, Masquerade, regulates cell-matrix interactions at the somatic-muscle attachment in the fly embryo, a process that also depends on integrin and laminin activity. It is speculated that a number of non-functional serine protease-like proteins, such as Masquerade and Scarf, are regulated temporally and spatially to promote the appropriate localization of BM proteins in a context-dependent manner to facilitate cell-matrix interactions and to ensure the maintenance of epithelial integrity. Similarly, among the large class of vertebrate functional and non-functional serine protease-like proteins involved in remodelling the extracellular matrix, some of them might exert a similar action (Sorrosal, 2010).
Search PubMed for articles about Drosophila Scarface
Bonin C. P. and Mann R. S. (2004). A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila. Genetics 167: 1801-1811. PubMed ID: 15342518
Rousset, R., et al. (2010). The Drosophila serine protease homologue Scarface regulates JNK signalling in a negative-feedback loop during epithelial morphogenesis Development 137(13): 2177-86. PubMed ID: 20530545
Sorrosal, G., Pérez, L., Herranz, H., and Milán, M. (2010). Scarface, a secreted serine protease-like protein, regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo. EMBO Rep. 11(5): 373-9. PubMed ID: 20379222
date revised: 30 December 2010
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