Gene name - Serrate
Cytological map position - 97F1-F2
Function - ligand for Notch
Keywords - Notch pathway
Symbol - Ser
Genetic map position - 3-92.5
Classification - TM protein - EGF family
Cellular location - cell surface
|Recent literature||Pandey, A. and Jafar-Nejad, H. (2018). Cell aggregation assays to evaluate the binding of the Drosophila Notch with trans-ligands and its inhibition by cis-ligands. J Vis Exp(131). PubMed ID: 29364239
Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. This study describes a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of >6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.
The function of Serrate in wing development demonstrates the importance of boundaries in morphogenesis and provides a direct example of how boundaries function. An important outcome of Serrate activity is the induction, through Notch, of the wing margin at the dorsal-ventral interface of the wing imaginal disc. Serrate is produced throughout the dorsal compartment of wing imaginal discs. The integrity of the dorsal compartment is maintained by cell adhesive molecules called integrins. Serrate functions downstream of fringe at the boundary between dorsal and ventral compartments (Kim, 1995). Loss of Serrate function in dorsal cells at the D/V boundary results in loss of the wing margin, and ectopic expression of Ser in both the dorsal and ventral compartment induces adult wing tissue outgrowth and wingless expression only in the ventral compartment.
Acting through the receptor Notch, Serrate induces the expression of wingless in cells of the adjacent ventral compartment (Speicher, 1994 and Kim, 1995).
Too much Serrate leads to disasterous consequences. Wing discs of Serrate Dominate, an allele that overexpresses Serrate, exhibit additional Serrate protein expression in the edge zone of the future wing margin. In these cells expression of wing margin specific genes, such as cut and wingless, are repressed (Thomas, 1995).
In contrast to Serrate, Delta is required in ventral cells at the dorsal/ventral compartment boundary (Doherty, 1996). Thus, two Notch ligands, Serrate in the dorsal compartment and Delta in the ventral compartment serve as Notch ligands, with Notch acting as receptor in both dorsal and ventral compartments.
Wingless is secreted and acts on downstream targets scalloped and vestigial to induce the cellular phenotype associated with the ventral fate, by way of an as yet unknown receptor. The fact that both Serrate and Delta are cell bound, and not secreted, insures that their effects at the boundary between dorsal and ventral compartments are felt only in adjacent cells across the dorsal/ventral boundary. This enhances the integrity of the boundary and accounts for very different cell fates on either side (Diaz-Benjumea, 1995).
Analysis of the function of Ser indicates that excess Ser can titrate out Notch function in the developing wing, an effect that is suppressed by an increase in the dosage of Notch. Since Serrate has been shown to bind Notch, this effect can be interpreted as an induction of a dominant negative activity of Serrate on Notch. Ubiquitous expression of Ser in the wing pouch throughout the development of the wing curtails wing development and produces wings that lack most of the margin and adjacent tissue (Klein, 1997).
Serrate can activate or inactivate Notch in a concentration-dependent manner as revealed by the expression of two targets of Notch activity: wingless and Enhancer of split. E(spl) is expressed in a stripe that straddles the DV interface at the beginning of the third larval instar. Ectopic expression of Ser product reduces the normal margin and produces two new margins on the ventral side of the developing wing. Ectopic expression of Ser in a Su(H) mutant has no effect on disc development or patterning and results in discs that are identical to those of Su(H) mutants. This demonstrates that the activity of Serrate during wing development requires Su(H). While the inactivation produced by ectopic Ser is likely to be mediated by a dominant negative effect over Notch, the activation is similar to that elicited by Delta and requires the product of the suppressor of Hairless gene (Klein, 1997).
Expression of Ser leads to smaller wings with thick veins. When wingless and Serrate are coexpressed, the resulting flies bear large wings that are covered with bristles. These wings have a different shape from those in wild-type: they lack a defined margin and are more round rather than elongated. This experiment shows that increased functional Wingless not only can suppress the dominant negative effect of Serrate expression, but can cooperate with Serrate to promote wing development. These wings are very similar to those that result from the expression of Delta and indicate that wingless enables Serrate to activate Notch. Coexpression of Ser and Notch generates very large wings, bigger than those that result from expression of Delta or coexpression of Serrate and wingless. These large wings have a clearly define margin with an antineurogenic phenotype. These results indicate that regulation of the concentration of Serrate during development must be an important way of regulating its activity (Klein, 1997).
Two different models are proposed. In one view the role of Serrate is to bind Notch at the DV interface to free Notch-bound Delta, which then would trigger events at the DV boundary that lead to wing outgrowth. This view is consistent with the observation of a dominant negative activity associated with Serrate and with the ability of Serrate to mimic Delta by activating Notch, leading to signaling through Su(H) and the consequent outgrowth of the wing. A different view sees Serrate as the active component of the signaling system, either alone or in combination with Wingless (Klein, 1997).
Diversification of Drosophila segmental morphologies requires the function of Hox transcription factors. However, little information is available that describes pathways through which Hox activities effect the discrete cellular changes that diversify segmental architecture. Serrate is a Hox gene target. Serrate acts in many segments as a component of such pathways. In the embryonic epidermis, Serrate is required for morphogenesis of normal abdominal denticle belts and maxillary mouth hooks, both Hox-dependent structures. The Hox genes Ultrabithorax and abdominal-A are required to activate an early stripe of Serrate transcription in abdominal segments. In the abdominal epidermis, Serrate promotes denticle diversity by precisely localizing a single cell stripe of rhomboid expression, which generates a source of EGF signal that is not produced in thoracic epidermis. In the head, Deformed is required to activate Serrate transcription in the maxillary segment, a region where Serrate is required for normal mouth hook morphogenesis. However, Serrate does not require rhomboid function in the maxillary segment, suggesting that the Hox-Serrate pathway to segment-specific morphogenesis can be linked to more than one downstream function (Wiellette, 1999).
A single allele of the Serrate gene (Ser5A29 ) has been isolated in a screen for mutations that enhanced hypomorphic phenotypes of mutations in the Hox gene Dfd. The Ser5A29 allele reduces the survival of Dfd hypomorphs to 40% of normal levels. To test whether this interaction with Dfd is allele-specific, null mutants of Ser were tested. The interaction strength of two such alleles with Dfd is 20-30% of the control chromosomes. Therefore, it is concluded that the function of Dfd is sensitive to the dose of wild-type Ser activity (Wiellette, 1999).
To explore the connection between Ser and Dfd functions, the embryonic/larval phenotypes of Ser mutants were examined. The Ser larval phenotype consists of blunt mouth hooks, anterior spiracle malformations and small imaginal discs in larvae of indeterminate age. Some animals lacking Ser die as embryos without emerging from the chorion, while others survive for variable periods as larvae. In both classes, the mouth hooks are blunt, lacking the sclerotic material that forms a curved hook in the normal structure. Since the mouth hook is a Dfd-dependent structure that is formed principally from maxillary cells, the requirement for Ser in normal mouth hook development is consistent with the genetic interaction between Ser and Dfd. Additionally, Ser mutant larvae have abnormally patterned abdominal denticle belts. Within each wild-type denticle belt from A2 to A8, the row 3 and 4 denticles have similar sizes and shapes but the hooks of row 3 point to the posterior, and those of row 4 point to the anterior. In Ser mutants, these two rows of denticles are combined into one row, the 3/4 row. The denticles in the 3/4 row have no regular polarity and about half of the embryos develop small denticles in this row. To determine whether the 3/4 row of denticles in Ser mutant larvae is a fusion of the two rows or a loss of one, denticles were counted, using the fourth abdominal segment (A4) as an example. The results are inconsistent with complete loss of either row 3 or row 4. Previous experiments have failed to detect any gross morphological defects in the cuticular features of embryos when Ser is ubiquitously expressed throughout Drosophila embryos. Using a Ser cDNA under Gal4-UAS control, the phenotypic effects of ectopic Ser were also assayed, focusing on denticle morphology. Ubiquitous expression of Ser induces no detectable changes in the shape, size or pattern of denticles. However, when Ser is ubiquitously expressed in a Ser heterozygous or null background, significant morphological defects are observed. The mouth hooks develop additional sclerotic material in the middle and base of the structure, and about a quarter of the embryos show duplication of mouth hook tips. Excessive sclerotic material also develops in the dorsal pouch. The expressivity of this phenotype varies from an extended and fragmented dorsal bridge to extreme sclerotization of the dorsal pouch and shortening of the lateralgräten. About half of the denticle belts in embryos of this genotype display the wild-type pattern but only rarely do embryos develop extra denticles around row 3. An additional result of ectopic Ser expression in a reduced-dosage background is excessive sclerotization of the proventriculus and gut. Both the dorsal pouch and the proventriculus are responsible for secretion of specialized cuticle to make head sclerites and gut lining, respectively (Wiellette, 1999).
Ser expression begins during embryonic stage 11, and eventually is transcribed in regions of the epidermis, tracheal trunks, foregut and hindgut, central nervous system and salivary ducts. Because of the phenotypic effect on mouth hooks, Ser expression was examined in the embryonic head. The transcript pattern in the head region at stage 12 includes the mandibular segment, the anterior and posterior lateral borders of the maxillary segment, and the anterior lateral and posterior ventral borders of the labial segment. A subset of cells in the clypeolabrum and dorsal head also accumulate Ser transcripts. The dependence of Ser expression on Dfd function and vice versa was tested and an epistatic relationship was found consistent with the genetic interaction. While there is no detectable change of Dfd expression in Ser mutants, Dfd and other head homeotic genes are required for the normal pattern of Ser transcription in the head. In Dfd mutant embryos, Ser transcripts are not expressed in cells in the anterior maxillary segment, which will eventually secrete part of the mouth hook. The posterior maxillary pattern is unchanged. When ectopic expression of Dfd protein is generated by heat shock (hs-Dfd), ectopic mouth hooks form in the labial and thoracic segments. These segments also express ectopic Ser on the lateral anterior and posterior borders. Thus, regulation of Ser by Dfd correlates with the segments where both normal and ectopic mouth hooks develop; it is concluded that Ser is one of the Dfd target genes that mediate mouth hook development. In Sex combs reduced mutant embryos, Ser transcripts are not expressed in the posterior maxillary segment or the anterior labial segment. capncollar mutant embryos, which develop ectopic mouth hooks from the mandibular segment, express Ser in the mandibular segment in a pattern similar to that of the maxillary segment (Wiellette, 1999).
Ser transcripts in the trunk are first detected at the extended germband stage in ventral patches in the middle of abdominal segments A2-A8 and in offset lateral patches. The ventral regions of thoracic segments do not exhibit Ser expression at this stage. As the germband retracts, the abdominal stripes intensify and develop sharp anterior borders. The first abdominal segment (A1) is unique: Ser expression begins later than in the other abdominal segments and forms a narrower stripe after germband retraction. After germband retraction, Ser transcripts can also be detected in the ventral regions of thoracic segments in broad, faint patches. Embryos mutant in all genes of the Bithorax Complex (BX-C), Ubx, abd-A and Abdominal-B (Abd-B), develop thoracic-type denticles throughout the trunk region. Consistent with this transformation, stage 11 and 12 BX-C mutant embryos have no Ser expression in ventral regions. Ventral Ser expression does begin in BX-C mutants after germband retraction, but the location and level of expression matches that of the thoracic segments. As expected, Ubx mutant embryos show a transformation of abdominal- to thoracic-type Ser expression only in A1; abd-A mutants show Ser transcript stripes in A2-A8 that are similar to the wild-type A1 pattern, and Abd-B mutants display no change in A1-A8 ventral Ser transcription. Thus Ubx function is sufficient to activate some Ser expression in the center of each segment, but abd-A function is required for the earlier, broader pattern of Ser transcription in A2-A8, a transcript pattern that correlates with complete diversification of denticle belts. Embryos lacking all trunk Hox functions express Ser at the margins of the anterior part of each trunk segment and at lower levels in the center of this region, a pattern almost the inverse of that seen in wild type. Transcription of Ser in the posterior-most region of each segment, probably corresponding to the posterior compartment, is completely suppressed. The delimitation of Ser expression to reiterated subsegmental stripes in the embryonic metameres suggests that segment polarity genes also regulate the Ser transcript pattern. ptc mutants lack ventral abdominal Ser transcripts, correlating with the loss of denticle diversity and number in ptc denticle belts. Ser transcription in wingless (wg) mutants appears in broad stripes, while hedgehog (hh) and engrailed (en) mutant embryos exhibit Ser transcription throughout almost the entire ventral epidermis of the abdominal segments. Broadened patterns of Ser transcription in these segment polarity mutants correspond to expanded fields of denticles that lack significant diversity of denticle type (Wiellette, 1999).
Serrate expression in segments is reflective of its function. The segmental boundary is persistently identified by engrailed expression in the posterior compartment of each segment, and, after germband retraction, by rho in the two most anterior cell rows of A2-A8. By stage 14, Ser transcripts are expressed in two to three rows of cells directly posterior to rhomboid-lacZ expression. The final position of Ser relative to En and rho-lacZ suggests that Ser is expressed in the cells that will produce denticle row 4 and those to the posterior. The anterior boundary of Ser expression appears to lie between the two rows of denticles that Ser affects phenotypically, suggesting that the function of Ser is limited to cells near its anterior boundary of expression. No changes of denticle pattern are found in fringe mutant embryos, indicating that fringe is not required to restrict Ser morphological function in the abdominal segments (Wiellette, 1999).
To understand how Ser affects denticle belt patterning at the single cell level, interactions of Ser with other genes that affect denticle diversity were investigated. In particular, the expression pattern of rho at the anterior of the Ser expression pattern made it a candidate for genetic interaction with Ser. The spitz-group gene rho is required for normal development of abdominal denticle rows 1 and 4. rho transcription first appears in a segmental pattern during stage 12, when it is activated in the anterior cells of each segment. Thoracic segments express rho in one row of cells at the anterior border of each segment, while ventral regions of segments A2-A8 express rho in two rows of cells, the posterior of which is dependent on Ubx/abd-A function. A1 develops rho expression in an intermediate pattern one to two cells wide. In a Ser null background, the abdomen-specific row of rho expression is missing and rho is transformed to the thoracic pattern in all abdominal segments. Stage 13 and early stage 14 Ser mutants show a row of unlabeled cells between Ser expression and the single row of rho expression, demonstrating that it is the posterior row of rho expression that is dependent on Ser. Lower levels of rho expression in Ser mutants suggest that anterior rho is partially dependent on Ser function, perhaps through the intervening posterior rho row. By late stage 14 in Ser5A29 mutant embryos, Ser transcripts have expanded anteriorly and are juxtaposed to the single row of rho. Consistent with this, Ser transcription is detected in an additional anterior row of cells in rho mutant embryos. Thus Ser is required for activation of rho in the posterior row of cells in the abdominal epidermis. Subsequently, rho is required to repress Ser within these same cells (Wiellette, 1999).
Denticle phenotypes show similarities between Ser and rho consistent with the observed regulatory interactions. A wild-type embryo produces six rows of denticles with each row identifiable by polarity and/or size. Ser mutants fail to separate rows 3 and 4, leaving five rows of denticles overall. In rho mutants, individual denticle rows are not as well defined, but it is possible to identify row 5 denticles in the middle of mutant denticle belts. Anterior to row 5 is a row that consists of small, stubby denticles. The most anterior row of denticles in the rho mutants contains sparse row 2 denticles. In rho,Ser double mutants, all of the denticles are similar to each other, and resemble a composite of type 5 and 2 denticles. The denticles are also disorganized, so that separate rows are not distinguishable. The difference between rho and rho,Ser double mutant phenotypes shows that the two anterior denticle rows in a rho mutant are still dependent on Ser for their diverse structures. This suggests that Ser function partially determines anterior denticle identity both within and anterior to its expression domain, independent of rho function. Additionally, the differences between Ser and rho,Ser double mutants show that a single row of rho expression is only partially sufficient to generate denticle diversity to its posterior. Thus the diversity of denticle rows 3 and 4 is dependent both on Ser regulation of rho and on independent Ser functions in denticle rows 3 and 4. Transcription of rho in maxillary segment cells is unchanged in Ser mutants, a result that correlates with wild-type mouth hooks in rho mutants and with the observation that rho,Ser and Ser mutants have identical mouth hook defects (Wiellette, 1999).
A model is presented for the roles of Ser, rho and Hox genes in the generation of denticle belt patterns in the thorax and abdomen. Three Hox genes (Antp, Ubx, and abdA) serve to establish the segmentally specific levels of Ser expression in the third abdominal segment and in the first two thoracic segments respectively. Ser is activated at stage 11 in abdominal parasegments by Ubx and abd-A functions but not in thoracic parasegments where Antp is the principal Hox function. Ubx function is required for the A1-type abdominal expression pattern of Ser, which is narrower and fainter than the pattern in other abdominal segments. This pattern correlates with a narrower, less complex denticle pattern in A1 than in more posterior segments. abd-A function is required for the wider, more abundant Ser stripes in A2-A8. Expression of Ser in the embryonic epidermis results in context-dependent responses, including rho expression, denticle belt patterning and normal development of the mouth hooks. These embryonic roles of Ser are apparently different from its roles in wing margin determination and wing outgrowth. One similarity is the short range over which Ser function is exerted, either at the anterior border of its ventral A2-A8 expression pattern, or at the dorsal/ventral margin of its expression boundary in the wing pouch. The spitz-group gene rho can potentiate Egfr activation via the Spitz (Spi) ligand. Egfr activation is required from late stage 11 to early stage 13 for patterning of the denticle belts, and rho, unlike spi and Egfr, has a spatially and temporally regulated expression pattern. Abdomen-specific rho expression is required for patterning of abdominal denticle rows 1 through 4, probably by allowing secretion of Spitz protein from denticle row 2 and 3 cells, which activates Egfr in neighboring cells. Ser function is required for activation of the abdomen-specific posterior row of rho transcription, expression of which is also dependent on Ubx/abd-A. The evidence presented in this paper suggests that Ser provides a critical intermediate that translates broad Hox and segment polarity domains into narrow stripes of rho expression, which then specify diversification at the single cell level. Ser and rho mutants each show only a single row of denticles between rows 2 and 5 of A2-A8, indicating that Ser and rho are both required for normal development of rows 3 and 4. rho,Ser double mutants develop row 5-like denticle identities throughout the denticle belt. Thus, either gene alone provides some A/P denticle diversity, while the double mutant lacks any diversity. If the only role of Ser were regulation of rho in denticle row 3 cells, then rho,Ser mutants should develop the same phenotype as rho mutants. Since this is not observed, it is concluded that Ser has identity functions independent of rho regulation. Ser function is required in the cells immediately to its anterior expression boundary and within the most anterior row of Ser-expressing cells; the effect within its own domain of expression may be a result of signaling from cells within the same row, or from those to the posterior (Wiellette, 1999 and references).
Ser function, presumably signaling through Notch, is required to determine the identity of the denticle row 3 cells, including activation of rho expression. Segmental rho, in turn, is one of the gene products required for localized Egfr activation in denticle row 4 cells. Thus denticle row 3 identity is dependent on Ser signaling and Rho function, while denticle row 4 identity depends on Ser function and on feedback from rho-expressing cells. This series of events suggests that abdominal Hox functions direct cellular diversification through the establishment of signaling centers. The limitation of Ser function to its anterior border generates a novel boundary within each segment. Activation of a single row of rho expression at this boundary then creates an additional signaling center in A2-A8, which controls the greater morphological diversity in A2-A8 epidermis. This use of a Notch ligand to produce a single cell signaling stripe may be a common theme: in dorsal/ventral wing margin patterning, Notch is activated in a single row of cells on either side of the margin in response to Delta and Ser boundaries, and Notch activation leads to a narrow, Wg-expressing, signaling center. Regulation of rho by the Notch pathway has been demonstrated in Drosophila wing vein patterning. However, in the larval and pupal wing primordia, Delta-activated Notch signaling results in repression of rho expression outside the forming vein. The difference in responsiveness of cells in the wing and the embryonic epidermis could be due to the identity of the signal (Ser versus Delta protein), or to additional identity factors present in the cell. rho, in turn, is required to maintain Delta expression in the provein region. Regulation of Notch function by Egfr activity has been described in C. elegans: translation of a C. elegans Notch homolog is downregulated in response to EGF signaling. Similar regulation may result from rho expression in the Drosophila embryonic epidermis, generating a feedback loop between Ser and rho as has been observed for Dl and rho in the forming wing (Wiellette, 1999 and references).
cDNA clone length - 5.6kb
Serrate is an integral membrane protein with an extracellular domain consisting of two cysteine-rich regions, one of which is organized in a tandem array of 14 EGF-like repeats (Thomas, 1991 and Fleming, 1990).
date revised: 28 January 98
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