Transcriptonal Regulation

Drosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. The ecdysone regulatory pathway controls wing morphogenesis and integrin expression during Drosophila metamorphosis. Mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion: this indicates a role for ecdysone in these morphogenetic responses. In some homo- and hetero-allelic crol combinations a few adult escapers can be recovered. All of these escapers also display wing defects. Fifty-seven percent of the adult escapers have held out wings with a partial (blister) or complete (balloon) separation of the dorsal and ventral wing surfaces, while the remaining 43% have either malformed or completely unfolded wings. The blisters in crol mutant wings are generally large and do not appear to have sharp boundaries. These phenotypes indicate a role for crol in cell adhesion and wing morphogenesis, raising the possibility that ecdysone signaling might play a role in regulating these processes. To test this hypothesis, the role of the ecdysone receptor in wing development was analyzed using the hypomorphic EcRk06210 allele. The wings of EcRk06210 homozygous mutants display cell adhesion and morphogenetic defects similar to those seen in crol mutants. At 18°C, 24% of the eclosed adults display venation defects, 32% have malformed wings, and 13% display wing blisters. The blisters are usually small and centrally located, although blistering of the entire wing (a balloon wing) can occasionally be seen. The most frequent venation defects include a small, extra vein that originates from the third longitudinal vein; an additional anterior crossvein, and a 'delta' thickening at the intersection between the posterior crossvein and the fourth longitudinal vein. crol and EcR mutations are shown to interact with mutations in genes encoding the integrin subunits. The frequency of blisters in if mutants is enhanced three- to four-fold by crol1 and crol2 and approximately sevenfold by crol3. In contrast, no interaction was observed between mysnj42 and crol1 or crol2, whereas the frequency of blisters and balloons in mysnj42 mutants is increased three- to four-fold by crol3, and the frequency of balloon wings increased approximately sevenfold (D'Avino, 2000).

alpha-Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of alpha- and beta-integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. The expression patterns of alphaPS1 and alphaPS2 in culture appear almost identical. After ~1 h of culture in the presence of ecdysone, alphaPS1 and alphaPS2 transcript levels decrease rapidly, becoming very low by 8 h after hormone addition. The alphaPS3 (scab) gene consists of two transcription units [Long-alphaPS3 (L-alphaPS3) and Short-alphaPS3 (S-alphaPS3)] that initiate from different start sites. Interestingly, L-alphaPS3 mRNA accumulates rapidly in response to ecdysone, peaking by 6-8 h after hormone addition, while S-alphaPS3 transcription appears unaffected by the hormone. Similar to S-alphaPS3, betaPS mRNA levels remain uniform throughout the time course. Thus, only alpha-integrin subunits are regulated by ecdysone in cultured larval organs, and they are either induced or repressed in response to the hormone (D'Avino, 2000).

These findings suggest that altered integrin gene expression in crol and EcR mutants lead to the defects observed in wing morphogenesis and cell adhesion. However, integrins also function in a wide range of other biological pathways during development, including tissue morphogenesis, cytoskeletal reorganization, memory, and gene expression. These widespread functions raise the possibility that ecdysone-regulated integrin expression may control multiple events during metamorphosis. For example, the if V2 semilethal allele displays a misshapen leg phenotype that resembles the defective legs seen in crol mutants, indicating that alphaPS2 functions may be recruited by the ecdysone pathway to regulate leg morphogenesis. Furthermore, since alphaPS3 has been proposed to mediate synaptic rearrangements, its ecdysone-induced expression in late third instar larvae may contribute to the extensive neuronal remodeling that occurs in the central nervous system during metamorphosis. Further studies of the tissue-specific functions of integrins during metamorphosis will provide a better understanding of how these critical cell surface receptors exert their multiple effects during development (D'Avino, 2000).

Protein Interactions

scab is a dimerization partner of Myospheroid (also known as betaPS). It is found as a 90kDa band associated with immunoprecipitates from an anti-betaPS antiserum. The 90 kDa protein forms a non-covalent, divalent cation-dependent complex with beta PS (Stark, 1997).


The larger transcript (4.9 kb) predominates in embryos, beginning with trace amounts at 0-2 hours, peaking at 8-11 hours, then decreasing through 16 hours of embryogenesis. It is strongly re-expressed during the pupal period and has some expression in the adult. A trace of the smaller transcript (4.5 kb) becomes evident by 6-8 hours of embryogenesis, increases in level from 8-16 hours and is the major form from 18 hours of embryogenesis through larval life. There is some increase in the expression of the larger transcript in late third instar larvae. The transcripts are approximately equal during pupal life and the smaller predominates in the adult. Adult males have a greater quantity of the larger transcript than do adult females, although the smaller transcript is more abundant in both (Stark, 1997).

During earliest embryogenesis, a low and uniform level of maternal Scab mRNA expression is seen; specific expression begins at the onset of gastrulation and continues throughout embryogenesis. The first discrete group of cells stained constitute the amnioproctodeal invagination, both prior to and during the invagination itself. In germ-band-extended embryos, signal is evident throughout the amnioserosa, being strongest at the junction with the epidermis. This continues through germ-band retraction and during dorsal closure. Subsequent to dorsal closure, strong expression is seen in the cells of the dorsal vessel. Two regions of expression are seen in the anterior of early germ-band-extended embryos in an unidentified region. These loose accumulations of cells appear to aggregate into two rounded clusters later. They are seen at either side of the foregut in stage 12, after germ-band retraction. The developing tracheal system shows alphaPS3 mRNA expression beginning in some of the cells of the invaginating tracheal pits. Expression continues in subsets of the tracheal system throughout embryogenesis, particularly in the transverse connectives and anterior spiracles. During stage 17, prominent expression is seen in the dorsal trunks as well as the transverse connectives and anterior spiracles. Portions of the midgut express Scab throughout development. Expression in early germ-band-extended embryos is in the presumptive visceral mesoderm. Both the anterior and posterior midgut primordia maintain strong expression, which continues through germ-band retraction and dorsal closure of the midgut. At these stages, the midgut expression appears restricted to the mesoderm. Subsequently, expression is strongest in the forming midgut constrictions and prefigures the appearance of any evident constriction. Some weak signal is seen in the forming proventriculus. By late in embryogenesis, signal is seen throughout the midgut, including the gastric caeca. Expression of alphaPS3 is seen in the midline cells of the ventral nerve cord at about stages 15 and 16. Lower level expression is seen in some cells scattered throughout the ventral nerve cord. RNA is also seen in some cells of the brain periphery at this time. Subsequent to dorsal closure, signal is evident in the antenno-maxillary complex, and this continues through late embryogenesis. A high level of signal is seen in the salivary glands beginning during their invagination and continuing throughout embryogenesis. Staining is present in both salivary ducts and the pore (Stark, 1997).

Integrins are necessary for the development and maintenance of the glial layers in the Drosophila peripheral nerve

Peripheral nerve development involves multiple classes of glia that cooperate to form overlapping glial layers paired with the deposition of a surrounding extracellular matrix (ECM). The formation of this tubular structure protects the ensheathed axons from physical and pathogenic damage and from changes in the ionic environment. Integrins, a major family of ECM receptors, play a number of roles in the development of myelinating Schwann cells, one class of glia ensheathing the peripheral nerves of vertebrates. However, the identity and the role of the integrin complexes utilized by the other classes of peripheral nerve glia have not been determined in any animal. This study shows that, in the peripheral nerves of Drosophila melanogaster, two integrin complexes (αPS2βPS and αPS3βPS) are expressed in the different glial layers and form adhesion complexes with integrin-linked kinase and Talin. Knockdown of the common beta subunit (βPS) using inducible RNAi in all glial cells results in lethality and glial defects. Analysis of integrin complex function in specific glial layers showed that loss of βPS in the outermost layer (the perineurial glia) results in a failure to wrap the nerve, a phenotype similar to that of Matrix metalloproteinase 2-mediated degradation of the ECM. Knockdown of βPS integrin in the innermost wrapping glia causes a loss of glial processes around axons. Together, these data suggest that integrins are employed in different glial layers to mediate the development and maintenance of the protective glial sheath in Drosophila peripheral nerves (Xie, 2011).

Peripheral nerves in vertebrates and Drosophila are organized in similar ways, with central axons wrapped by an inner class of glia that are surrounded in turn by layers of external glia. The glia and the surrounding ECM establish a tubular sheath to protect axons from physical damage and pathogens. This study shows that at least two integrin heterodimers are expressed in these different glial layers and are localized at focal adhesions with Ilk and Talin. αPS2βPS integrin is prevalent in the PG and the αPS3βPS integrin is more prevalent in the wrapping glia (WG). Since βPS2 integrin is expressed mostly in the outermost PG and can bind ligands that contain the tripeptide RGD sequence, it most likely functions by binding to ECM ligands in the NL. The majority of αPS3 integrin is expressed by the internal SPG and WG and αPS3 integrin has been shown to interact with laminins in Drosophila. However, it is possible that the integrin complex in the WG might have other ligands and might mediate direct cell-cell interactions, since a pronounced basal lamina associated with the peripheral axons was not detected and Mmp2 expression had no effect in the internal regions of the peripheral nerves (Xie, 2011).

Peripherial glia (PG) form the outermost glial layer in the Drosophila nervous system but their origin and function are not well understood, even though they were identified some time ago. Drosophila PG are structurally similar to their vertebrate counterparts and have been proposed to have similar roles. Vertebrate perineurial cells and the associated collagen fibers provide important mechanical support to nerves and their development might rely on ECM-mediated signals, as β1 integrin is found in the perineurium. However, little is known about the role of integrins in perineurial cells (Xie, 2011).

The current results show that Drosophila PG express αPS2 and βPS integrin subunits (and to a lesser extent αPS3), which colocalize with both Ilk and Talin. Knocking down integrins disrupts perineurial wrapping, but it was not possible to distinguish whether the PG failed to initiate wrapping or failed to maintain their processes around the nerves to accommodate the growing nerve surface. However, degradation of the neural lamella (NL) by Mmp2 overexpression generates a PG wrapping phenotype similar to that of βPS RNAi. Thus, binding of βPS integrin to ligands in the ECM mediates the radial spread of PG around the tubular structure of the nerve and suggests that PG retract their membrane when integrin-ECM interaction is interrupted. Moreover, knockdown of Talin in the PG produces a similar wrapping defect. This suggests that, in the PG, integrin adhesion complexes mediate the connection between the extracellular NL and the intracellular actin cytoskeleton and are required for the initiation or maintenance of glial ensheathment (Xie, 2011).

The function of the PG in the peripheral nerve is not well understood. The PG do not generate an impermeable barrier and it is the subperineurial glia (SPG) layer that creates the blood-nerve barrier. Loss of PG ensheathment did not result in paralysis or lethality, which are signs of a disrupted blood-brain barrier. Larger molecules (~500 kDa) are blocked by the NL or the PG, perhaps mirroring the protective function of the vertebrate perineurium against pathogens. However, in Mmp2-overexpressing larvae, the affected nerves become thin and are difficult to retain intact during tissue preparation. This suggests that the NL and PG provide important mechanical support as is seen with the perineurium in mammalian nerves (Xie, 2011).

In the center of Drosophila peripheral nerves, the WG embed axons in bundles or individually within single membrane wraps, similar to the non-myelinating Schwann cells of vertebrate peripheral nerves. The current results show that WG predominantly express αPS3 and βPS integrin subunits in complexes positive for Ilk and Talin along the WG membrane. When βPS expression is knocked down, the complexity of the glial processes between the associated axons is greatly reduced. Only a few long processes and small membrane protrusions are observed around the axons, suggesting that the WG might be retracting their processes in the absence of βPS integrin. The role of integrins appears to be conserved between WG and Schwann cells in vertebrates. For example, myelinating Schwann cells lacking β1 integrin or Ilk do not extend membrane processes around axons, resulting in impaired radial sorting. The role of integrins in non-myelinating Schwann cells is not known but the current results suggest that integrins have similar functions in Drosophila and vertebrates in mediating the glial ensheathment of peripheral axons (Xie, 2011).

No clear ECM has been observed in the internal regions of Drosophila nerves by immunofluorescence analysis or transmission electron microscopy. Therefore, the integrin complex could promote WG sheath formation by mediating direct cell-cell adhesion between the glial membrane and its associated axon, or between glial membranes. A potential candidate for an integrin-interacting protein expressed on axons or glia is Neuroglian, the Drosophila L1 (Nrcam) homolog. L1 is an Ig domain transmembrane protein that is known to bind RGD-dependent integrins. Loss of the integrin-binding domain of L1 results in wrapping defects in both myelinating and non-myelinating Schwann cells. However, the presence of low levels of ECM cannot be ruled out given the weak laminin immunolabeling that was observed. Even though Mmp2 expression in the WG had no effect, it is still possible that the integrin-mediated adhesion is Mmp2 resistant. To resolve this issue, further ultrastructural and genetic studies will be required (Xie, 2011).

Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila

Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. This study shows that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the alpha-integrin subunits including alphaPS1 encoded by mew and alphaPS3 encoded by scb, and the beta-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a prerequisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). These studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations (Lin, 2013).

Effects of mutation or deletion

Southern blot analysis of heterozygous scab adults using the complete alpha probe reveals DNA polymorphisms in 6 alleles. Mutant embryos homozygous for l19 or 1035 alleles of scab, sometimes undergo a secondary closure after the initial failure of dorsal closure. Unlike myospheroid (mys) mutant embryos, whose muscles detach from the body wall, scb embryos show vigorous muscular movements of the cuticle; some of those undergoing secondary dorsal closure actually do hatch. In addition to dorsal closure, another phenotype observed in embryos lacking betaPS, but not observed in embryos null for alphaPS1, alphaPS2 or both, is a twisting of the germ band. Time-lapsed videomicroscopy reveals that during germ-band extension in scb X5, scb X6, and scb 2 embryos, and in embryos deficient for scb, the germ band twists laterally rather than extending dorsally, as in wild-type embryos, so that the ventral side of the posterior midgut is visible from the lateral side of the embryo. As in myospheroid mutant embryos, proper orientation of the germ band is recovered by the completion of germ-band extension (Stark, 1997).

The strong expression of Scab in the dorsal vessel led to an examination of scab embryos for defects in this tissue. Defects in the dorsal vessel have not been reported for myospheroid mutant embryos, although a detachment of alary muscles from the heart (posterior dorsal vessel) and its failure to mature at late stages has been identified in embryos lacking zygotic, but not maternal betaPS. myospheroid mutant embryos lacking both maternal and zygotic betaPS were examined to preclude the possibility of maternal rescue. The heart and dorsal vessel form from two types of cells: the external pericardial cells and the internal cardioblasts. myospheroid minus, scb X5, scb X6, scb 2 and scb-deficient embryos were all stained with antibodies that recognize pericardial cells. Embryos from scb and deficiency lines show mislocalization of the pericardial cells, which normally organize in a line along the edge of the dorsal cuticle, and appear to have fewer of these cells in this area than wild type at the same stage. A similar (although more severe) defect could be seen in mys- embryos: the pericardial cells appear to dissociate, migrate randomly and are sparse. The increase in severity of the defect suggests the possibility that either alphaPS1 or alphaPS2 may function in this process as well. The defect in mys- is also strikingly similar to that published for laminin A mutations (Yarnitzky, 1995).

Laminin is a common ligand for integrins in vertebrates. Since Scab mRNA is expressed in parts of the trachea throughout embryogenesis, antibodies to examine the trachea in wild-type, mys-, scb X5, scb X6, scb 2 and both scb deficiencies. To explore further the potential of laminin as a ligand, lamA 9-32 mutant embryos were also examined for tracheal defects. Embryos from each of the mutant stocks have significant gaps in the dorsal trunk of the trachea, which are not present in wild-type embryos. Due to the strong expression of alphaPS3 mRNA in the salivary glands throughout development, the glands of wild-type, mys - , scb X5 , scb X6 and scb 2 embryos were examined using a reporter gene. mys and scb embryos frequently show one gland to be misshapen and smaller than the other. The salivary gland is sometimes shifted closer to the midline. In conclusion, several defects are seen in scb mutant embryos, all of which are shared with mys and some with lamA; among them are defects previously reported in mys, but not in multiple edematous wings (aPS1) or inflated (aPS2) mutations. Noteworthy is the fact that these defects arise in areas where Scab is strongly expressed (Stark, 1997).

Cell migration during embryogenesis involves two populations of cells: the migrating cells and the underlying cells that provide the substratum for migration. The formation of the Drosophila larval midgut involves the migration of the primordial midgut cells along a visceral mesoderm substratum. Integrin adhesion receptors are required in both populations of cells for normal rates of migration. In the absence of the PS integrins, the visceral mesoderm is disorganized, the primordial midgut cells do not display their normal motile appearance and their migration is delayed by 2 hours. Removing PS integrin function from the visceral mesoderm alone results in visceral mesoderm disorganization, but only causes a modest delay in migration and does not affect the appearance of the migrating cells. Removing PS integrin function from the migrating cells causes as severe a delay in migration as the complete loss of PS integrin function. The functions of PS1 and PS2 are specific in the two tissues, endoderm and mesoderm, since one cannot substitute for the other. In addition, there is a partial redundancy in the function of the two PS integrins expressed in the endoderm: PS1 (alphaPS1betaPS) and PS3 (alphaPS3betaPS: alpha PS3 is Scab), since loss of just one alpha subunit in the midgut results in either a modest delay (alphaPS1) or no effect (alphaPS3). Thus, the migration of primordial midgut cells along a substratum provided by the visceral mesoderm requires the coordinated function of all three PS integrins. The PS2 integrin is required in the visceral mesoderm for the formation of an optimal substratum for migration. The PS1 and PS3 integrins are required in the endodermal cells for their migration over the visceral mesoderm. These two integrins are partially redundant in the midgut, but PS3 cannot fully replace PS1 integrin function. The roles of small GTPases in promoting migration of the primordial midgut cells were also examined. Dominant negative versions of Rac and Cdc42 cause a very similar defect in migration as does the loss of integrins, while dominant negative versions of Rho and Ras have no effect. Thus integrins are involved in mediating migration by creating an optimal substratum for adhesion, adhering to that substratum and possibly by activating Rac and Cdc42 (Martin-Bermudo, 1999).

The separate loss of either alphaPS1 or alphaPS2 does not produce the same migration phenotype as loss of betaPS. However, loss of the PS2 integrin does produce the same defects in the organization of the visceral mesoderm as loss of all PS integrins. There are at least two possible explanations for this result: (1) either the alphaPS subunit is sufficient to mediate migration, or (2) there is another a subunit that can partially compensate for the loss of alphaPS1 or alphaPS2. In order to distinguish between these possibilities, embryos mutant for both alphaPS1 and alphaPS2 were examined. If the first explanation were correct then one would expect the double mutant phenotype to be as strong as the loss of betaPS. Endodermal cells from alphaPS1alphaPS2 minus embryos show a greater delay in cell migration than that observed in the single alpha subunit mutants. This can be seen when only one or two cells are found in contact in the middle in the double mutant embryos at a time when several cells have met in the single mutant embryos. Nevertheless, in this double mutant combination, the endodermal cells send projections and the delay in cell migration (1 hour) is still less severe than that observed in betaPS minus embryos. This situation is different from the role played by the integrins in muscle attachment, where the loss of PS1 and PS2 integrins is equivalent to the loss of all PS integrins. Therefore, these results suggest that additional alpha subunits exist that can compensate for the loss of alphaPS1 and alphaPS2. These alpha subunits could be required in the endoderm, in the visceral mesoderm or in both tissues (Martin-Bermudo, 1999).

Since loss of PS2 appears to affect the formation of the visceral mesoderm substratum as strongly as loss of the betaPS subunit, and this does not substantially delay migration, a model is favored in which other alpha subunits are required in the endoderm. Since alphaPS3 (Scab) is also expressed in the endoderm, it is an obvious candidate for a second alpha required in the endoderm for cell migration. Therefore, as the next step in analyzing the role of integrins in midgut migration, the specific requirements of the betaPS subunit in the two different tissues was examined, as was the role of alphaPS3 (Martin-Bermudo, 1999).

Analysis of embryos mutant for alphaPS3 supports previous evidence indicating that there is no defect in midgut migration, showing that the PS3 integrin is not essential for midgut migration. However, it is possible that there is some redundancy between the two alpha subunits expressed in the endoderm, so that alphaPS1 compensates for the lack of alphaPS3 and vice versa. To test this, embryos lacking both PS1 and PS3 integrins were generated and midgut migration was examined. Because alphaPS1 and alphaPS3 genes are on different chromosomes, it was difficult to use the GAL4 system to express any marker, so an enhancer trap line, 258, was used that is expressed in the endodermal cells as they migrate. This is a nuclear marker that allows identification of the position of the endodermal cells and therefore the monitoring of their migration, although it does not reveal the morphology of the cells. In the absence of both alphaPS1 and alphaPS3 subunits, the defect in the migration of the midgut is as strong as in the absence of the betaPS subunit, consisting of a delay of approximately 2 hours. Embryos doubly mutant for alphaPS3 and alphaPS2 subunits, were examined and no difference in midgut development was found as compared to the loss of alphaPS2: this shows that there is no overlap in function of the PS2 and PS3 integrins. In conclusion, the main requirement for PS integrins during migration is provided by the combination of PS1 and PS3 integrins. To investigate whether PS3 is required to regulate gene expression in the midgut, the patterns of expressions of the two integrin target genes were examined. Loss of PS3 function does not cause any change in their expression. Thus, PS1 function in migration can also be partially performed by the PS3 integrin (there is a small delay in the absence of PS1), while PS1 function in the regulating gene expression or adhesion to the visceral mesoderm cannot be performed by PS3 (Martin-Bermudo, 1999).

A new olfactory conditioning procedure is described using short training trials with discrete presentation of conditioned stimuli (CS) and unconditioned stimuli (US). A short odor presentation along with a single-shock stimulus produces modest but reliable and reproducible learning. Multiple trials present sequentially improved performance with increasing trial number. Trial spacing has a significant impact on performance. Two trials presented with a short intertrial interval (ITI) produces no improvement over a single trial; two trials with a 15 min ITI significantly boosts performance. This effect requirs two associative trials, because substituting one of the trials with the CS alone, US alone, or an unpaired CS-US, fails to boost performance. The increase in initial performance with two trials decays within 15 min after training. Thus, the effect is short-lived. The utility of using a battery of tests, including a single short trial, two massed trials, and two spaced trials, to investigate parameters of memory formation in several mutants was demonstrated (Beck, 2000).

To investigate the utility of the short program in examining parameters of olfactory learning and memory, several memory mutants along with controls were tested in a simple battery consisting of one trial, two massed trials, and two spaced trials. This provides a rapid way of quantitating initial performance with very discrete CS and US presentations and for probing the normal enhancement of learning in spaced over massed training. Three different parameters of olfactory conditioning were examined and these were compared with long program training. (1) It was determined whether observed differences between memory mutants could be observed using the short program. (2) Some memory mutants have been rescued in long program conditioning using heat-inducible transgenes. It was of interest to see whether behavioral rescue would be revealed after short program conditioning. (3) Many memory mutants of Drosophila are dominant for memory formation. It was therefore of interest to see in whether dominance-recessivity relationships would be the same after short program conditioning (Beck, 2000).

The mutant rutabaga (rut) was initially isolated from genetic screens for defects in operant olfactory conditioning, and the gene was subsequently shown to encode an adenylyl cyclase. The allele used here, rut2080, contains a P-factor at the locus and exhibits poor performance in long program olfactory classical conditioning. Volado (Vol), also known as Scab, is a short-term memory mutant with a defective alpha-integrin gene. Vol1, like rut2080, contains a P-factor at the locus and disrupts the expression of one of two RNAs (Vol-l, 4.6 kb) from the locus, eliminating the alpha-integrin gene product, VOL-L, encoded by this RNA. A second allele, Vol2, contains a small deletion of the locus that selectively eliminates the alternative RNA transcript (Vol-s, 4.4 kb) and the alternative alpha-integrin isoform, VOL-S, encoded by this RNA. Both of these alleles show a dominant deficiency in 3 min and 15 min memory after training in the long program for olfactory classical conditioning. In addition, they both exhibit a performance decay that parallels that of the control at approximately the 50% level over a time course of 3 hr after conditioning. Behaviorally, therefore, they appear identical after long program conditioning. This led to the suggestion that the two alpha-integrin isoforms encode redundant functions. However, it is also possible that the failure to detect behavioral differences between the alleles is caused by an insensitivity of long program conditioning. Alternatively, the long program might obscure authentic differences (Beck, 2000).

Single-trial, short program training of rut2080, Vol1, and Vol2 shows that all mutants perform poorly immediately after conditioning. In addition, they exhibit behavioral differences. The rut2080 and Vol1 mutants are sensitive to two-trial, spaced conditioning over two-trial, massed conditioning. In this respect, both mutants are qualitatively like control animals. This phenotype is consistent with the interpretation that the mutants are defective in memory formation, or acquisition, a conclusion made for Vol1 from several other behavioral tests. The Vol2 mutant's performance is strikingly different. Single-trial training fails to induce any conditioning whatsoever! Therefore, all three memory mutants show defective performance after short program training. In addition, the assay detects differences between Vol1 and Vol2 that are not detected by long program conditioning (Beck, 2000).

A test was perfomed to see whether short program training with massed and spaced variations would be sensitive to behavioral rescue experiments using transgenes expressed from the heat shock (HS) promoter. The performance deficit of Vol 2 after long program conditioning can be rescued to control levels by induced expression of the Vol-s RNA, if HS is provided 3 hr before training. Control ry flies, Vol2 mutants, and Vol2 mutants carrying the transgene (Vol2, hspVol-s) were given HS or no HS 3 hr before single-trial training, two massed trials, or two spaced trials. Immediate performance after training was measured (Beck, 2000).

As before, spaced training leads to enhanced performance of ry controls, which is independent of any HS treatment. Similarly, Vol2 mutants (HS or no HS) and transgenic animals (Vol2, hspVol-s; no HS) perform at naive levels after single-trial training and show only modest performance after two massed or two spaced training trials. Most importantly, HS of Vol2, hspVol-s flies rescues the Vol2 behavioral phenotype to wild type qualitatively and quantitatively. Single-trial performance after HS is indistinguishable from controls, as is the failure of two massed trials to build significantly on single-trial performance. Two spaced trials given after HS produce performance equivalent to the control animals. This leads to two important conclusions: (1) the short program can be used to evaluate behavioral rescue with conditional transgenes; (2) the Vol2 mutant is rescued by the conditional transgene not only after long program conditioning but also after short program training. Thus, the transgene appears to provide all essential functions for rescue in both behavioral assays (Beck, 2000).

The short program also proves to be a valuable assay for probing the dominance and recessivity of memory mutants. The Vol1 and Vol2 alleles are both dominant for performance after long program training when tested at 3 min and 15 min after conditioning. These alleles were retested, along with a new allele, Vol3, a 925 bp deletion that simultaneously removes both the Vol-l and Vol-s transcription units. These alleles offer the opportunity to eliminate one copy of the Vol-l transcription unit (Vol1/+), one copy of the Vol-s transcription unit (Vol2/+), both copies of Vol-l (Vol1), both copies of Vol-s (Vol2), one copy each of Vol-l and Vol-s (Vol3/+ and Vol1/Vol2), and both copies of Vol-s along with one copy of Vol-l (Vol3/Vol2). The different genotypes were trained with a single trial, two massed trials, or two spaced trials (Beck, 2000).

In contrast to long program conditioning, the Vol1 allele is completely recessive after short program conditioning. This indicates that a single copy of the Vol-l transcription unit is sufficient for normal behavior in this assay. Furthermore, the behavior of Vol1 homozygotes is reduced overall but appears qualitatively like the control by showing the normal enhancement because of spaced training. These data suggest that the VOL-L integrin is not an essential factor for conditioning but modulates the effectiveness of the conditioning (Beck, 2000).

The Vol2 allele is partially dominant for performance after single-trial and two-trial massed conditioning (Vol2/+ performance). However, at least one copy of the Vol-s transcription unit is required for any conditioning to occur after a single trial, because genotypes that remove both copies of the Vol-s transcription unit (Vol2 and Vol3/Vol2) exhibit absolutely no conditioning whatsoever! Thus, the VOL-S integrin is an essential factor for single-trial conditioning. Furthermore, Vol2 is completely dominant for the memory enhancement that comes with spaced training, because no effect of spaced training is observed in genotypes that remove one or both copies of the Vol-s transcription unit (Vol2, Vol2/+, Vol1/Vol2, Vol3/+, and Vol3/Vol2). The Vol2 allele, therefore, has a stronger effect overall on conditioning than the Vol1 allele. These behavioral genetic data suggest that the two integrin isoforms play qualitatively different roles in the processes underlying memory formation, a conclusion suggested by other behavioral and physiological studies (Beck, 2000).

One complication of long program training is that the rapid shock pulses (12, with 1 per 5 sec) alters the salience of the odor cues used for training, as assayed by the normal avoidance of the aversive odor cues. This effect has been ascribed to stress produced by the shock pulses. In addition, some learning mutants respond more acutely than controls to this stressful situation, producing the worrisome situation that odor cues during testing may be more salient to controls than to certain memory mutants (Beck, 2000).

The effects of shock pulses delivered in mock training trials of the short program for a single trial, two massed trials, or two spaced trials were assayed. Electric shock was delivered normally in the mock training, but odor cues were replaced by fresh air. Afterwards, the odor avoidance was measured and compared with animals receiving no pretreatment. The ry control and the rut2080 mutant avoid the two odors used for training to the same extent when given a choice of fresh air in a T-maze. Electric shock pulses delivered according to the time schedule for training with a single trial, two massed trials, or two spaced trials do not alter subsequent avoidance of the odors. Similar results have been obtained for Vol1 and Vol2. Thus, short program training eliminates the confounding factor of stress and odor desensitization that occurs with long program olfactory classical conditioning (Beck, 2000).

These data illustrate that (1) the short program generates highly reproducible behavior; (2) the discrete stimuli used by the program provide a method for dissecting the rate of memory formation, or acquisition. Multiple trials presented sequentially can be used to compare the memory formed in controls and mutants after one, two, or three trials, for example, to obtain measurements of the rate of memory formation. This is impossible with the long program because the training protocol induces ceiling levels of performance after a single trial. (3) Memory stability can be measured immediately after single-trial training to survey the state of very early memory and its subsequent decay. (4) The unique behavioral enhancement after two spaced trials over single-trial conditioning or two massed trials offers another criterion by which to compare mutants and controls. As shown here, certain mutants (Vol1 and Vol2) differ dramatically in their responses to this training. This may provide insights into the mechanisms that operate to produce the enhancement, as well as help to dissect the roles of various gene products in memory formation. (5) The short program circumvents several problems associated with long program training. The spacing of shock pulses during long program training may produce some extinction during periods when the CS is present but the US is not. The intense shock pulses are thought to produce a stressful situation that alters the salience of odors. The salience change may be different from strain to strain, yielding a situation that may produce misinterpretations about their relative behavior. Nevertheless, long program conditioning will not be replaced by the short program procedure, because the examination of longer-term memory requires the induction of high levels of initial performance. It is believed that experiments using both schedules may yield the most meaningful information about each mutant (Beck, 2000).

Volado, the gene encoding the Drosophila alphaPS3-integrin, is required for normal short-term memory formation, this fact supports a role for integrins in synaptic modulation mechanisms. The Volado protein (Vol/Scab) is localized to central and peripheral larval Drosophila synapses. Vol-L isoform is strongly concentrated in a subpopulation of synaptic boutons in the CNS neuropil and to a variable subset of synaptic boutons at neuromuscular junctions (NMJs). Mutant morphological and functional synaptic phenotypes were analyzed at the NMJ. Volado mutant synaptic arbors are structurally enlarged, suggesting Vol negatively regulates developmental synaptic sprouting and growth. Mutant NMJs exhibit abnormally large evoked synaptic currents and reduced Ca2+ dependence of transmission. Strikingly, multiple forms of Ca2+- and activity-dependent synaptic plasticity are either reduced or absent. Conditional Volado expression in mutant larvae largely rescues normal transmission and plasticity. Pharmacologicially disrupting integrin function at normal NMJs phenocopies features of mutant transmission and plasticity within 30-60 min, demonstrating that integrins acutely regulate functional transmission. These results provide direct evidence that Volado regulates functional synaptic plasticity processes and support recent findings implicating integrins in rapid changes in synaptic efficacy and in memory formation (Rohrbough, 2000).

Vol protein is present at low levels throughout most larval synaptic terminals in fixed preparations. In contrast, staining is strongly localized to a limited and variable subpopulation of central and peripheral synapses. Localized punctate Vol expression is present extensively in the CNS neuropil, contained within the broader expression domains of the constitutive presynaptic proteins Synaptotagmin (Syt) and Synaptobrevin (Syb). When transgenically expressed in a neuronal subset [using the gal4 (4G) driver], Syt-GFP and Syb-GFP are observed to be concentrated in extensive puncta and varicosities resembling Vol-localized expression, strongly supporting the conclusion that Vol is indeed concentrated at central synaptic boutons. Likewise, at the NMJ localized Vol expression is observed at all classes of terminals and at all morphological classes of synaptic boutons. Thus, Vol is clearly expressed in a variable population of central and peripheral synaptic boutons (Rohrbough, 2000).

There are several alternative ways to interpret the intriguing and unusual pattern of synaptic Vol expression. In one hypothesis, Vol may be present only transiently, may be restricted to an extremely limited population of synapses, or both. However, the significant morphological and functional phenotypes exhibited at mutant NMJs are inconsistent with Vol functioning only at a limited subset of boutons or synapses. A second hypothesis that is consistent with the morphological and physiological data is that Vol is expressed at most or all NMJs, but it is concentrated at detectable levels at a subset of terminals and in a variable number of boutons at a given time. One possibility is that Vol is transiently concentrated to distinct boutons in response to an inducing signal. Although there is no direct evidence for such dynamic relocalization, it is consistent with previous integrin studies and all of the data presented in this study. A third possibility, which cannot presently be excluded, is that an unknown mechanism that causes epitope modification or masking of the antibody recognition site creates variability in the level of Vol immunostaining. The more extensive localization of the protein to the central neuropil likely reflects the high density of synaptic connections in this region or differences in the dynamic regulation of localized expression by activity or other mechanisms. However, the observation of Vol expression in variable subsets of synaptic boutons is consistent in both central and peripheral synaptic terminals (Rohrbough, 2000).

Vol mutant NMJs exhibit moderate but significant overgrowth at multiple terminal types, suggesting Vol has a broad developmental role in limiting morphological synaptic growth. The altered evoked transmission amplitudes and defective plasticity properties in Vol mutants, however, indicate that Vol has an additional functional role regulating synaptic transmission and activity-dependent synaptic modulation. The homozygous Vol1 and Vol2 strains selectively eliminate expression of the Vol-l and Vol-s integrin isoforms, respectively (Grotewiel, 1998). The severity of transmission phenotypes among both viable and lethal mutant (Vol1, Vol3, Vol4) animals again strongly suggests that Vol directly regulates function throughout the synapse, or alternatively, is able to indirectly influence the function of a broader area via some signal transduction pathway (Rohrbough, 2000).

Both viable (Vol1) and lethal Vol mutant alleles (Vol3, Vol4) display abnormally elevated evoked transmission amplitudes, altered Ca2+ dependence of transmission, and severe defects in short-term plasticity at the NMJ. In the viable Vol2 memory mutant, basal transmission amplitude and short-term plasticity are essentially normal. However, all alleles, including Vol2, display significantly reduced post-tetanic potentiation (PTP), indicating that mutant plasticity defects do not result simply from elevated basal transmission levels. It should be stated clearly that these forms of synaptic plasticity at the NMJ, although highly conserved at central synapses, cannot be directly correlated to particular phases of behavioral learning or memory. The Vol mutant defects are consistent, however, with an inability to rapidly modulate transmission in response to increased presynaptic activity and to maintain changes in transmission strength in the absence of maintained input plasticity properties; these properties are likely to be relevant for memory formation (Rohrbough, 2000).

Conditional expression of the Vol-s isoform in the lethal Vol4 allele dramatically rescues transmission amplitude, short-term facilitation (STF), augmentation, and PTP to levels near or equal to those at control NMJs. Similar results were obtained by driving Vol expression over most of larval development, or over ~36 hr in late larval stages. The subtle differences in the completeness of rescue of individual transmission properties may indicate different sensitivities to the timing and level of Vol expression relative to normal, or different functions for the Vol-l and Vol-s isoforms. These results strongly support a direct role for Vol in synaptic transmission and plasticity mechanisms. Whereas the pharmacological perturbation with a RGD integrin inhibitory peptide (Gly-Arg-Gly-Asp-Ser-Pro) lacks the specificity of the Volado genetic knock-out and transgenic rescue approach, it offers the only available means of assessing the consequence of acutely disrupting normal synaptic integrin function. At wild-type NMJs, exposure to RGD rapidly (within 30-60 min) leads to increased transmission amplitude and loss of presynaptic short-term facilitation. The similarity of the RGD-dependent modulation of transmission to the Vol mutant phenotype suggests Vol may mediate this rapid modulation. Consistent with this possibility, the alphaVol sequence includes three consensus extracellular Ca2+-binding domains conserved in integrin RGD receptors. However, the ligand interactions of Vol are not yet characterized. These results also indicate that the RGD-dependent modulation may be mediated in part by other synaptic integrins. The most likely such candidate is alphaPS2/betaPS, which is abundantly localized to type I boutons at the NMJ and has been shown to mediate RGD-dependent cell adhesion in vitro. These results primarily demonstrate that acutely altering synaptic integrin function in Drosophila, as in vertebrates, can rapidly and dramatically modulate transmission efficacy (Rohrbough, 2000).

Integrin interactions at the synapse stabilize basal transmission, mediate rapid (minutes) changes in transmission strength, and are required for normal Ca2+- and activity-dependent forms of plasticity. The cellular mechanisms by which Vol regulates synaptic transmission and memory formation and the significance of its variable pattern of synaptic localization remain to be determined. The richness of known integrin interactions suggests multiple potential roles for Vol in synaptic mechanisms. One possibility is that alphaVol, with its betaPS integrin partner, is linked to or positioned to interact with the synaptic vesicle fusion machinery. Integrins have recently been reported to be localized to active zones of neuromuscular synapses. Such a close relationship with presynaptic Ca2+ channels and the Ca2+-sensitive vesicle fusion machinery could position integrins to directly modulate the probability of transmitter release in response to Ca2+ influx, thereby regulating both evoked release and rapid modulatory processes. Disrupting this relationship in Volado mutants might account for increased excitatory junctional currents (EJC) amplitudes, reduced Ca2+ dependence of transmission, loss of short-term forms of Ca2+-dependent plasticity, and the rapid phenocopying of similar synaptic defects by the RGD peptide (Rohrbough, 2000).

For synaptic integrins to contribute to long-term changes in synaptic efficacy, however, interactions with additional cellular signaling pathways would appear necessary. Memory and long-term experimental forms of synaptic plasticity are known to require Ca2+- and cAMP-dependent signaling, and both appear to be accompanied by prolonged functional modulation of transmission properties, and/or the physical stabilization or addition of synaptic connections. Dynamic changes in the structure and function of synaptic connections could potentially be regulated by altered integrin adhesion and signaling function, triggered by increased synaptic activity. Localized integrin expression is detected in hippocampal dendritic spines and postsynaptic densities, and appearance of new dendritic spines has been documented within minutes after LTP induction, suggesting integrins could be used in formation or stabilization of new synapses or structural changes to existing contacts. Transient changes in cytosolic Ca2+ levels are known to regulate subcellular integrin redistribution and cell morphology. Increased neuronal activity and synaptic Ca2+ influx may trigger Vol redistribution and concentration in synaptic boutons (Rohrbough, 2000).

Several modulatory signaling pathways downstream of activity-dependent increases in synaptic Ca2+ could potentially regulate dynamic changes in synaptic integrin localization and adhesion, including the cAMP-, protein kinase C (PKC)-, and Ca2+/calmodulin (CaM)-dependent pathways, and calcineurin. These transduction pathways are implicated in synaptic modulation and memory formation in Drosophila and mammalian systems. Integrin-ligand binding can in turn trigger Ca2+ influx and release from intracellular stores, increased PKC activity, and activation of the presynaptic Ca2+-binding protein calreticulin, which interacts dynamically with alpha-integrins to regulate adhesion and transient Ca2+ influx. Interestingly, calreticulin is increased after long-term sensitization in Aplysia and has been proposed to modulate gene expression after integrin activation, suggesting that integrin-mediated forms of long-term plasticity and memory may be associated with new gene expression necessary to stabilize or 'cement' transient morphological alterations (Rohrbough, 2000).

Integrins are concentrated within growth cones, but their contribution to axon extension and pathfinding is unclear. Genetic lesion of individual integrins does not stop growth cone extension or motility, but does increase axon defasciculation and axon tract displacement. In this study, a dosage-dependent phenotypic interaction is documented between genes for the integrins, their ligands, and the midline growth cone repellent, Slit, but not for the midline attractant, Netrin. Longitudinal tract axons in Drosophila embryos doubly heterozygous for slit and an integrin gene, encoding alphaPS1, alphaPS2, alphaPS3, or ßPS1, take ectopic trajectories across the midline of the CNS. Drosophila doubly heterozygous for slit and the genes encoding the integrin ligands Laminin A and Tiggrin reveal similar errors in midline axon guidance. It is proposed that the strength of adhesive signaling from integrins influences the threshold of response by growth cones to repellent axon guidance cues (Stevens, 2002).

Axon fasciculation and guidance were studied in the CNS of embryos mutant for the ß integrin gene myospheroid (mys) and three alpha integrins, alphaPS1 (mew), alphaPS2 (if), and alphaPS3/4 (scb). It is not known whether the scb locus affects alphaPS3 or alphaPS4 or both, because the alphaPS4 locus is separated from alphaPS3 by 259 bp. Variation in penetrance of mutant phenotype was observed with all integrin alleles. The three longitudinal fascicles are intact in loss-of-function mutations in mys, which encodes the only ß integrin expressed in the CNS; however, midline fusions of the most medial tract are seen in some segments. Axon tract structure is least disrupted in mew mutant embryos. No midline guidance errors are seen; however, the longitudinal fascicles appeared to be thinner, with occasional defasciculation. if mutant embryos are similar in phenotype to mew, but also reveal a low frequency of midline crossover errors. Midline fusions as well as transient merging of lateral fascicles are seen in scb mutant embryos (Stevens, 2002).

A scb,sli recombinant could not be isolated, in order to assess the scb,sli/scb phenotype. These genes would be expected to recombine in 1 of 25 chromosomes; however, no recombinants were isolated in 200 chromosomes screened. It was possible to investigate the interaction of these genes by using overlapping deficiencies. Midline guidance was assessed in scb and sli heterozygotes, in trans to a deficiency that uncovers either scb or sli or both genes. Consistent with the characterization of the sli and scb phenotypes, sli in trans to a deficiency uncovering sli has complete midline fusion of all axon tracts, and scb in trans to a deficiency uncovering only scb has an integrin mutant phenotype. The semidominant interaction of sli and scb is also confirmed when scb is trans to a deficiency uncovering only slit or when sli is trans to a deficiency uncovering only scb. A synthetic scb homozygote and sli heterozygote phenotype was generated in embryos with a scb mutant allele in trans to a deficiency uncovering both scb and sli. This phenotype is qualitatively similar to the scb/sli phenotype, revealing frequent midline crossing or fusion of the two most medial but not the most lateral axon fascicles (Stevens, 2002).

The midline axon phenotype of integrin mutants is part of a more complex phenotype involving defasciculation, irregular fascicle position, and 'wavy' axon trajectories. This phenotype emerges even when slit function is normal and may reflect axon guidance functions of known integrin ligands in the nervous system. Two integrin ligands have been identified within the LT: Laminin, and Tiggrin. The Fas II phenotypes of embryos mutant for these ECM proteins were characterized to clarify their possible contribution to axon guidance (Stevens, 2002).

Scab and wing development

Morphogenesis of the Drosophila wing depends on a series of cell-cell and cell-extracellular matrix interactions. During pupal wing development, two secreted proteins, encoded by the short gastrulation (sog) and decapentaplegic (dpp) genes, vie to position wing veins in the center of broad provein territories. Expression of the Bmp4 homolog dpp in vein cells is counteracted by expression of the secreted Bmp antagonist sog in intervein cells, which results in the formation of straight veins of precise width. A screen was performed for genetic interactions between sog and genes encoding a variety of extracellular components and interactions were uncovered between sog and myospheroid (mys), multiple edematous wing (mew) and scab (scb), which encode ßPS, alphaPS1 and alphaPS3 integrin subunits, respectively. Clonal analysis reveals that integrin mutations affect the trajectory of veins inside the provein domain and/or their width and that misexpression of sog can alter the behavior of cells in such clones. In addition, a low molecular weight form of Sog protein has been shown to bind to alphaPS1ßPS. Sog can diffuse from its intervein site of production into adjacent provein domains, but only on the dorsal surface of the wing, where Sog interacts functionally with integrins. Finally, it has been shown that Sog diffusion into pro-vein regions and the reticular pattern of extracellular Sog distribution in wild-type wings requires mys and mew function. It is proposed that integrins act by binding and possibly regulating the activity/availability of different forms of Sog during pupal development through an adhesion independent mechanism (Araujo, 2003).

sog mRNA is confined to intervein cells during pupal development. Since Sog protein diffuses during early embryonic development, however, it was of interest to see whether Sog might also travel from its intervein site of production into the provein region during pupal development. Pupal wings were stained with the anti-Sog antiserum and a dynamic pattern of Sog protein distribution was observed, including vein competent domains as well as intervein cells. Anti-Sog staining is initially patchy (around 20 hours apf), stronger on the dorsal surface and mostly restricted to intervein cells. Shortly thereafter (22-26 hours apf), Sog staining spreads into provein cells on the dorsal surface of the wing, at which point it is excluded only from the most central vein-proper cells. On the corresponding ventral surface, however, Sog staining remains excluded from the entire provein region throughout pupal development (i.e., up to 34 hours apf). Between 26 and 30 hours apf, Sog staining fills all the provein domains on the dorsal surface, with increased levels of staining observed at the provein/intervein border. At 30 hours apf, Sog staining diminishes overall and becomes restricted to intervein cells and hemocytes running in the middle of the vein. Since sog mRNA is detectable only in intervein cells during the examined pupal period, it is concluded that Sog protein must be delivered to cells within the provein territory on the dorsal surface by some form of passive diffusion or active transport (Araujo, 2003).

Because diffusion of Sog into provein domains is restricted to the dorsal surface of the wing where integrins interact with Sog, it was asked whether integrins play a role in regulating the distribution of Sog protein on the dorsal surface of pupal wings. Marked mys minus or mew minus clones were generated in an otherwise wild-type background and Sog staining was examined. In control wings, double-labeling with anti-ß integrin and anti-Sog antisera confirmed that the dorsally restricted pattern of reticular Sog staining extends beyond ß-integrin staining into provein domains. By contrast, Sog staining has a patchy intracellular appearance in dorsal mys minus clones, and is excluded from wild-type provein cells on the dorsal surface that are adjacent to mys minus clones. In such cases where mys minus clones are located on one side of a provein domain, Sog is still able to enter the provein region from the opposite mys+ side of the same vein. These results demonstrate that mys is required for diffusion or transport of Sog into the vein competent domain. Consistent with the observation that only dorsally located integrin minus clones can alter the course of veins, it was found that only dorsal mys minus clones modify Sog distribution in the pupal wing. Similarly altered Sog staining was observed within mew minus clones on the dorsal wing surface resulting in punctate rather than reticular staining and lack of Sog diffusion into the provein region. These results demonstrate that the ßPS and alphaPS1 integrins play an important role in determining the distribution of Sog protein in the pupal wing (Araujo, 2003).

In this study, three primary lines of evidence are provided that integrins play an important role in regulating Bmp signaling in provein regions of the pupal wing: (1) integrin minus clones generated on the dorsal surface of the wing alter the trajectory and/or width of adjacent veins; (2) a truncated form of Sog present in pupal wings binds to alphaPS1; (3) diffusion of Sog into provein domains, which is restricted to the dorsal surface of the wing, depends on integrin function. Cumulatively, these results strongly suggest that the ability of Sog to diffuse or to be transported into provein regions on the dorsal surface depends on an interaction with integrins (Araujo, 2003).

Consistent with Sog interacting genetically with integrins to alter the course of veins on the dorsal surface of the wing, it was found that the alphaPS1 and ßPS-integrins are required for the diffusion or transport of Sog from dorsal intervein cells where sog mRNA is expressed into adjacent provein regions. Since alphaPS1 binds Sog, this physical interaction may contribute to regulating the distribution of Sog. The 8B anti-Sog antiserum used in this study, that recognizes Sog protein in intervein cells and inside the provein domain, detects an epitope located near the second cystein repeat (CR2). Consequently, Sog fragments that diffuse or that are delivered to provein cells must be either full length, which weakly binds to alphaPS1 in co-immunoprecipitation experiments, or fragments that contain CR2. The truncated Supersog-like fragment that binds strongly to alphaPS1 in coimmunoprecipitation experiments should not be recognized by the 8B antiserum. Therefore, integrins may differentially regulate the distribution of Sog fragments on the dorsal surface of the pupal wing, restraining the movement of broad spectrum Bmp inhibitory Sog fragments (such as Supersog-like molecules) and allowing or mediating transport of other fragments to provein cells, such as full-length Sog, which also has a vein inhibitory function. Unfortunately, it is not possible currently to examine the diffusion of Supersog-like fragments directly because the 8A antiserum is not suitable for staining pupal wings. These findings suggest that integrins regulate the delivery or diffusion of active Sog protein from intervein cells into the vein competent domain. In contrast to the dorsally restricted functions of integrins required for vein development, the adhesive functions of integrins depends on subunits functioning on both surfaces of the wing (Araujo, 2003).

There are several possible mechanisms by which interaction with integrins could modulate Sog activity in pupal wings. Elevated sog expression results in vein truncation, while misexpression of dpp induces ectopic veins, indicating that sog restricts vein formation by opposing Bmp signals emanating from the center of the vein (Yu, 1996). One possibility is that such a Bmp inhibitory form(s) of Sog must interact with integrins in order to diffuse or be transported into provein domains (on the dorsal surface of the wing only). This hypothesis would be consistent with the finding that veins appear to be attracted to integrin minus clones. Such a vein repulsive form(s) of Sog would presumably act as a Bmp antagonist (Araujo, 2003).

According to the simple model in which integrins are essential for delivering a Bmp inhibitory form of Sog to provein cells, one would expect that integrin minus and sog minus clones would generate similar phenotypes in which veins deviated towards the mutant clones and/or broadened within them. However, sog minus clones induce meandering of veins (Yu, 1996) -- veins show only a weak tendency to track along the outside of sog minus clones, in contrast to integrin minus clones, which bend or widen veins in a more dramatic fashion. One possible explanation for the differences between the sog minus and integrin minus phenotypes is that there are several different endogenous forms of Sog in pupal wings, which might exert opposing activities. If multiple Sog fragments exert effects on vein development, some providing repulsive and others attractive activities on vein formation, the differences between the behaviors of sog- and integrin- clones could be explained by a repulsive (Bmp inhibitory) form(s) of Sog selectively requiring an interaction with integrins. The possibility that a positive Bmp-promoting activity of Sog might also be present that acts as a vein attractant has precedent in that a positive Sog activity has been proposed to explain a requirement for Sog in activating expression of the Dpp target gene race in early embryos. Structure/function studies of Sog have also revealed a potential Bmp promoting form of Sog, which is longer than Supersog forms. According to this model, altering the balance between repulsive and attractive Sog activities would generate different vein phenotypes. In the total absence of sog, both repulsive and attractive activities would be lost, generating a mild meandering vein phenotype in which neither attraction nor repulsion clearly dominates, as is observed in sog minus clones (Yu, 1996). If an interaction with integrins were required only for production or delivery of Bmp inhibitory forms of Sog into the vein, then integrin minus clones, which still contain the Bmp-activating forms of Sog, could exert a net attractive influence on veins, leading to more pronounced deviation of veins toward the clones. This hypothesis is consistent with vein phenotypes observed associated with integrin minus clones that cross over veins or run along both sides of the vein, such as narrowing, bending and wandering of veins; these phenotypes are similar to those observed in correspondingly located sog minus clones. The existence of different Sog fragments bearing opposing activities would also explain the different phenotypes obtained upon ectopic Sog expression in some sogEP lines (Yu, 1996), such as sporadic ectopic vein material between L3 and L2 and meandering L2 veins in addition to vein loss in other areas (Araujo, 2003).

Another possible explanation for the differences between the sog minus and integrin minus phenotypes is that integrins may regulate the activity of extracellular signals in addition to Sog. One hint of such an activity is that when a scb minus clone falls within the provein area, the vein splits around the border of the clone in a cell autonomous fashion. Since this later phenotype is enhanced by ectopic sog expression in veins (e.g., in a sogEP background), alphaPS3 may normally promote Bmp signaling within the vein. Although the identity of such potential targets is unknown, candidates would include Bmps (e.g. Dpp or Gbb) or Bmp receptors. Further analysis will be needed to explain the basis for the different behaviors of sog minus and integrin minus clones, as well as the variations observed in different integrin minus clones (Araujo, 2003).

In summary, it is proposed that Sog fragments with differential activities may regulate vein formation. The vein bending phenotype observed in the absence of alphaPS1 would result from a remaining attractive Sog activity that outweighs the activity of a repulsive form of Sog, which can no longer be delivered from intervein cells. Since ßPS integrin forms heterodimers with both alphaPS1 and alphaPS3 mys would be expected to be required for the activity of both alphaPS chains. Consistent with this expectation, the phenotype of mys minus clones (i.e., broad poorly defined veins) resembles a hybrid of those observed for mew minus and scb minus clones (Araujo, 2003).

Endocytosis has been shown to play an important role in the establishment of Bmp activity gradients. The endocytic pathway has been implicated in transport of Dpp between cells by transcytosis during larval wing development. During early embryonic development, formation of a Sog protein gradient in dorsal regions also relies on the action of Dynamin, although in this preblastoderm context, it has been proposed that endocytosis limits the dorsal diffusion of Sog, which is essential for the partitioning of the dorsal ectoderm into epidermis and amnioserosa. Vertebrate alpha3ß Integrin (E. deRobertis, personal communication to Araujo, 2003) has been shown to bind to the Xenopus Sog counterpart Chordin in vitro, leading to endocytosis of Chordin (Araujo, 2003).

Although the possible regulation of Sog endocytosis by integrins is not addressed in this current study, the altered distribution of Sog within integrin minus clones is suggestive of such a role. Reticular Sog staining, which outlines the cell perimeter is lost in integrin minus clones on the dorsal surface, leaving only a punctate intracellular staining. This mis-localization of Sog implicates integrins in internalizing and/or trafficking of Sog to the cell surface. Because appropriately located integrin minus clones also block the accumulation of Sog in adjacent pro-vein domains, the observed defects in Sog distribution between the surface and the cytoplasm may underlie the failure to deliver Sog to vein competent cells. The endocytic pathway could promote the transport of Sog to pro-vein cells by a mechanism similar to that proposed to be involved in the transport of Dpp along the AP axis during larval stages. Alternatively, endocytosis could function to limit Sog diffusion as is the case during embryogenesis. According to this latter scenario, integrins would normally prevent or reduce Sog endocytosis because integrins are necessary for delivery of Sog to pro-vein cells. Integrins have been shown to play a direct role in endocytosis of viral particles and in mediating membrane traffic through the endocytic cycle. Indirect mechanisms for integrin-mediated endocytosis may also exist that would not involve endocytosis of the integrin receptor itself, but of other components that regulate Sog trafficking. Further analysis will be necessary to investigate whether Drosophila integrins regulate delivery of Sog to endocytic vesicles or transport of Sog through the endocytic pathway to adjacent cells (Araujo, 2003).

The modulatory effect of integrins on Sog activity described in this paper are likely to be mediated by dpp and/or gbb signaling because existing evidence indicates that Sog is a dedicated modulator of Bmp signaling. In addition, the phenocritical period for mys and sog interaction coincides with that for interaction between sog and dpp (Yu, 1996). The existence cannot be excluded of an additional role of integrins in regulating vein formation through another pathway, such as the Egf and Notch pathways, which have been shown to exert important roles on vein development. However, the integrin minus clonal phenotypes described in this manuscript are observed only on the dorsal surface and all known components of the Egfr pathway promote vein development on both surfaces of the wing (Araujo, 2003).

mysnj42 and scb1 were found suppress the thickened vein phenotype of tkv1 mutants, which raises the possibility of a direct interaction between integrins and a Bmp receptor involved in wing vein development. The vein splitting and vein thickening scb minus clonal phenotypes are reminiscent of tkv mutant phenotypes, which derive from a positive requirement for Bmp signaling for vein formation inside the vein competent domain and a negative ligand titrating function that limits the range of Bmp diffusion into the intervein territory adjacent to the provein domain. The fact that scb is expressed in both vein and intervein territories is consistent with a dual action of scb. Additional experiments will be necessary to investigate whether scb plays a direct role in modulating Bmp receptor activity (Araujo, 2003).

Diffusion of putative growth factors and the shaping of their activity gradients have been the focus of intense interest since Allan Turing formulated the concept of morphogens (Turing, 1952: see 'The chemical basis of morphogenesis'. Philos. Trans. R. Soc. Lond. B Biol. Soc. 237: 37-72). Recently, several groups have described mechanisms to explain how soluble factors can create morphogen gradients. These include: (1) degradative proteolysis and a retrieval role for endocytosis in creating the early embryonic Sog gradient; (2) regulated endocytosis of wingless extracellular transport of Wg in membrane bound argosomes; (3) planar transcytosis (as is required for Dpp movement in the wing imaginal disc), and (4) the formation of thin cell extensions (cytonemes) that deliver Dpp over several rows of cells. Protein-protein interactions in the extracellular milieu, such as those described here, may also be capable of modulating the magnitude and spatial pattern of Bmp activity, working independently or in conjunction with other mechanisms (Araujo, 2003).

Genetic screen in Drosophila melanogaster uncovers a novel set of genes required for embryonic epithelial repair

The wound healing response is an essential mechanism to maintain the integrity of epithelia and protect all organisms from the surrounding milieu. In the 'purse-string' mechanism of wound closure, an injured epithelial sheet cinches its hole closed via an intercellular contractile actomyosin cable. This process is conserved across species and utilized by both embryonic as well as adult tissues, but remains poorly understood at the cellular level. In an effort to identify new players involved in purse-string wound closure a wounding strategy suitable for screening large numbers of Drosophila embryos was developed. Using this methodology, wound healing defects were observed in Jun-related antigen (encoding DJUN) and scab (encoding Drosophila alphaPS3 integrin) mutants and a forward genetics screen was performed on the basis of insertional mutagenesis by transposons that led to the identification of 30 lethal insertional mutants with defects in embryonic epithelia repair. One of the mutants identified is an insertion in the karst locus, which encodes Drosophila betaHeavy-spectrin. betaHeavy-spectrin (betaH) localizes to the wound edges where it presumably exerts an essential function to bring the wound to normal closure (Campos, 2010).

Using previously described DC or wound healing mutants a pilot screen was performed to validate the embryonic wounding strategy. The fact that a member of the DJNK pathway (Jra/DJun) was identified in the assay is in accordance with other reports that implicate this pathway in wound healing. Specifically, two mutations in components of the DJNK pathway, bsk/DJNK and kay/DFos, were previously shown to have defects in fly larval and adult wound closure, respectively. In addition, a reporter construct has been describes that requires consensus binding sites for the JUN/FOS complex to be activated upon wounding. Interestingly, treporter activation was still observed in Jra mutants, which suggests that additional signaling pathways are involved in wound closure (Campos, 2010).

An apparent discrepancy arose when the assay revealed a phenotype with Jra but not with puc mutants, another component of the same signaling pathway. This result might be explained by the fact that Jra and puc function in opposite directions in the DJNK signaling pathway. Puc functions as a pathway repressor, so in a puc mutant the JNK pathway should be less repressed and an opposite effect to a Jra mutation could be expected. In addition, activation of a puc-lacZ reporter has been shown to occur in larvae, wing imaginal discs, and adult wounds that take 18-24 hr to close, but it is only robustly detectable 4-6 hr postpuncture. Embryonic wounds are faster to heal, and even after inflicting a large laser wound on stage 14/15 embryos, no activation of the puc-lacZ reporter (assessed in open wounds 3 hr postwounding by immunofluorescence; data not shown) was detected. This observation suggests that, in rapidly healing epithelial wounds, the JNK pathway is not activated to high enough levels to trigger auto-inhibition (Campos, 2010).

The α-integrin scab was never before implicated in embryonic wound healing, but this mutant's phenotype comes as no great surprise. The first scab mutation was isolated due to its abnormal larval cuticle patterning. The scab gene encodes for Drosophila α-PS3 integrin, which is zygotically expressed in embryonic tissues undergoing invagination, tissue movement, and morphogenesis. Integrin proteins are involved in cell-matrix interactions and α-PS3 integrin regulation, in particular, mediates zipping of opposing epithelial sheets during DC. Similarly, the observation of a wound defect in scb5J38 mutants is consistent with a role for α-PS3 integrin in zipping of opposing epithelial cells during the healing process (Campos, 2010).

A previous study using confocal video microscopy has shown that Rho11B mutants take twice as long to close an epithelial wound when compared to wild type. Rho1 was confirmed in the assay to be important for wound healing, although with a weaker phenotype (22% of embryos had unclosed holes). This result shows nonetheless that the assay can be sensitive enough to pick up a 'weak' wound healing mutant such as Rho11B, which is still able to heal wounds albeit slower than wild type (Campos, 2010).

The genes identified in the screen represent a variety of functions indicating that wound healing is a complex mechanism that requires the participation of many cellular processes. A large class of the candidate mutants are involved in several aspects of gene expression, including factors that regulate chromatin remodeling (dUtx and Pc), elongation (dEaf), splicing (Glo and CG3294), and translation (CG33123). These factors are likely needed during wound healing for the induction of a repair transcriptome. Interestingly, JNK signaling-dependent Pc group (PcG) gene downregulation has been observed during imaginal disc regeneration. In addition, a recent study revealed that PcG methylases are downregulated during wound healing, while counteracting demethylases, Utx and Jmjd3, are upregulated. The results for the Pc and Utx mutants are consistent with these studies and highlight the importance of epigenetic reprogramming in the repair process (Campos, 2010).

Some of the genes such as arc-p20 and karst probably have a more direct role in the cell shape changes that drive the tissue morphogenetic movements during epithelial repair. The gene product of arc-p20 is a component of Arp2/3, a complex that controls the formation of actin filaments, and karst encodes a component of the spectrin membrane cytoskeleton. Also related to morphogenesis, CG12913 encodes an enzyme involved in the synthesis of chondroitin sulfate, which is usually found attached to proteins as part of a proteoglycan, suggesting a predictable contribution of the extracellular matrix in the tissue movements necessary for wound healing (Campos, 2010).

The epithelium is the first line of defense of the organism against pathogens and tissue integrity. It would thus seem plausible that genes involved in innate immunity could be identified with the screening protocol. Indeed, two of the genes (Ser12 and CG5198) seem to point to the involvement of the immune response in the healing of the laser-induced wounds. Ser12 is a member of the serine protease family, a class of proteins that has been shown to play a role in innate immunity. The CG5198 gene has no described function in Drosophila so far, but its homolog, CD2-binding protein 2, is involved in T lymphocyte activation and pre-RNA splicing. Another candidate that might represent a link to immunity is Atg2, a gene important for the regulation of autophagy, a process by which cells degrade cytoplasmic components in response to starvation. In Drosophila, autophagy has been linked to the control of cell growth, cell death, and, recently, to the innate immune response mechanism against vesicular stomatitis virus and listeria infection (Campos, 2010).

Isolation of an insertion in the stam gene points to the involvement of the JAK-STAT signaling cascade in this regenerative process. Interestingly, stam has been shown to be involved in Drosophila tracheal cell migration and is upregulated following Drosophila larvae infection by Pseudomonas entomophila (Campos, 2010).

One candidate could be involved in the uptake or export of some important wound signal (CG7627) as this gene encodes for a multidrug resistant protein (MRP), part of the ABC transporter superfamily, involved in drug exclusion properties of the Drosophila blood-brain barrier (Campos, 2010).

The kinase encoded by grapes is the Drosophila homolog of human Check1 (Chk1) involved in the DNA damage and mitotic spindle checkpoints. All the Chk1 literature has focused on its role during the cell cycle. However, the Drosophila late embryonic epithelium is a quiescent tissue, even after wounding. Understanding Grapes function in this context is a challenging task that could lead to new paradigms. One hypothesis is that Grapes is involved in tension sensing, as it is in the spindle checkpoint, or may uncover a cellular repair process that could help damaged cells 'decide' to either die by apoptosis or participate in the repair process (Campos, 2010).

The remaining genes with a putative function represent a wide range of general metabolic processes (aralar1, gs1-like, CG4389, CG9249, CG11089, and CG16833), suggesting that healing the epithelium is a highly demanding process (Campos, 2010).

Finally, a significant number of genes that have not yet been studied and do not contain identifiable protein domains (CG2813, CG31805, CG6005, CG6750, CG10217, CG15170, and CG30010) were selected. At the moment it is not possible to predict the role that these genes may play, but further study may help to identify novel wound healing regulatory mechanisms (Campos, 2010).

One of the mutants identified in the transposon screen was kstd11183, an insertion in the βH-spectrin locus. This mutation is likely producing a truncated protein terminating three amino acids into the P-element insertion. Other mutations identified in nearby segments 14 (kst14.1, kst2) and 16 (kst1) lead to the production of a detectable truncated protein so it is likely that karstd11183 mutation also gives rise to a truncated protein. These mutant forms of βH lack approximately half of the wild-type protein, including a COOH-terminal PH domain region, which is involved in targeting the protein to the membrane, thus producing a potential dominant negative form of βH. However, the karstd11183 mutant should still have maternally loaded wild-type protein, as previous studies describe a complete absence of maternal protein only by the third instar larval stage. This maternal contribution is likely the main reason that this mutant, as well as the other mutants isolated in the screen, does not have a fully penetrant wound healing phenotype (Campos, 2010).

βH-spectrin was shown to localize to the actomyosin purse string, a supracellular contractile cable that forms rapidly upon wound induction. Live imaging has demonstrated that actin and myosin can accumulate in this cable structure within minutes after wounding. Unfortunately, due to the size of the βH gene (>13 kb) cloning and tagging it for live imaging is not possible using standard methods, but the experiments in fixed tissue reveal that βH can accumulate very rapidly in this cable structure. βH accumulation was observed at the earliest time point technically feasible, 15 min postwounding. These observations are consistent with previous studies, also in fixed tissue, demonstrating rapid changes in βH localization during the process of cellularization in Drosophila embryos. Taken together, it is clear that at least the βH component of the membrane skeleton is not just a static structural scaffold as the name implies, but rather a dynamic protein capable of responding to or directing changes in cellular dynamics. The studies suggest that polarized redistribution of βH exerts an essential function to facilitate actin-based cellular responses, such as cable accumulation/maintenance and wound edge filopodia dynamics, which are necessary to properly close a wound (Campos, 2010).

βH has been previously observed in association with actin 'rings' during development of Drosophila and C. elegans. Arguably, C. elegans provides an example of actin ring function most analogous to the Drosophila wound edge purse string. During the final stages of C. elegans development, cortical arrays of actin in the outer epithelial cells, the hypodermis, dramatically reorganize to form parallel apically localized bundles of circumferencial supracellular actin rings. In this system, sma1, the C. elegans ortholog of βH, also localizes apically to these actin rings. In sma1 mutants the rings fail to productively contract and begin to disorganize, losing connection to the cell membranes. An additional phenotype observed in these mutants is the inability of cells to change their shape, a process normally 'directed' by these contractile rings, the end result being a short worm, a phenotype seen as functionally analogous to an unclosed wound in the Drosophila system (Campos, 2010).

In Drosophila, βH has been implicated in modulating cell shape changes during apical constriction of follicle cells (a process also involving actin rings) and has been proposed to function as a link between cross-linked actin networks/rings and the cell membrane. Further studies revealed that the C-terminal domain of βH has the ability to directly modulate the apical membrane area by regulating endocytosis, adding one more tantalizing piece of evidence pointing to the fact that βH could be a major player in cell shape changes, not only as a structural link but also by directly modulating the membrane area in response to cytoskeletal clues (or vice versa) (Campos, 2010).

Although it is known from previous studies that the actin cable is not absolutely required for wound closure, the process takes much longer without one. In Rho1 mutant embryos, cells lacking a cable are able to pull the wound closed using filopodia. The filopodial defect observed in karst mutants, adds another line of evidence to the absolute requirement of these structures for wound closure. In addition to the reduced actin cable accumulation and filopodial dynamics in karst mutants (which would lack the C-terminal domain responsible for membrane modulation), a lack of cell shape change is seen in the wound edge cells. Taken together, these data and the published work, introduce the intriguing possibility that βH could be serving as a link between wound edge dynamics and the coordinated cell shape changes usually observed in wild-type wound edge cells. The combination of the proposed ability of βH to modulate the apical membrane area as well as cross-link actin and act as an apical membranewide scaffold for other interactions, makes βH a good candidate to provide the physical link that would coordinate tissuewide actions, such as supracellular actin cable contraction, with the individual cellular responses, such as cell shape change and polarized filopodia activity (Campos, 2010).


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scab: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 October 2013 

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