Laminin A
Anterior-posterior axis formation in the Drosophila oocyte requires activation of the EGF receptor (EGFR) pathway in the posterior follicle cells (PFC), where it also redirects them from the default anterior to the posterior cell fate. The relationship between EGFR activity in the PFC and oocyte polarity is unclear, because no EGFR-induced changes in the PFC have been observed that subsequently affect oocyte polarity. This study shows that an extracellular matrix receptor, Dystroglycan, is down-regulated in the PFC by EGFR signaling, and this down-regulation is necessary for proper localization of posterior polarity determinants in the oocyte. Failure to down-regulate Dystroglycan disrupts apicobasal polarity in the PFC, which includes mislocalization of the extracellular matrix component Laminin. These data indicate that Dystroglycan links EGFR-induced repression of the anterior follicle cell fate and anterior-posterior polarity formation in the oocyte (Poulton, 2006; full text of article).
This study has identified DG as a gene whose expression pattern is both regulated by EGFR signaling in the PFC and necessary for oocyte polarity. These findings provide a mechanistic link between EGFR activity in the PFC and polarization of the oocyte. Furthermore, it was discovered that defects in apicobasal polarity caused by ectopic DG also are present in the PFC where EGFR signaling is disrupted, possibly due to the misexpression of DG in these cells. In addition, the findings that ectopic DG leads to mislocalizations of Lan at the apical surface of the PFC indicates a process of cell–cell communication in which EGFR-regulated DG expression in the PFC controls Lan organization in the ECM that in turn may affect localization of posterior determinants in the oocyte (Poulton, 2006).
It was reported that loss of LanA in the PFC disrupts oocyte polarity, which seems to be in conflict with the suggestion that high levels of apical Lan in the PFC perturbs oocyte polarity. However, a model in which Lan is required in early oogenesis, but must be localized basally after EGFR activation and DG down-regulation, reconciles these findings. In the previous research on loss-of-function lanA mosaic egg chambers, oocyte polarity defects observed at stage 9/10 could be generated only by larger lanA PFC clones. Because follicle cells are only mitotically active until stage 6/7 of oogenesis, these large PFC clones present at stage 9/10 would have represented sizeable lanA clones in prestage-6 follicle cells. Because Lan is present on the apical surface of these pre-PFCs, the polarity defects observed at stage 9/10 may have resulted from perturbation of some earlier Lan-dependent processes, such as organizing receptors on the facing surfaces of the oocyte or follicle cells. Consistent with this model, the addition of Lan to myotubes in culture is sufficient to organize the receptors integrin and DG, as well as their respective cytoplasmic counterparts, vinculin and dystrophin. Alternatively, it could be that the role of Lan in mediating the relationship between the PFC and oocyte is sensitive to any disruption of the ECM stemming from either the loss or misexpression of Lan, which then is sufficient to negatively affect oocyte polarity. Either of these models demonstrates the importance of the ECM in this process and ultimately may lead to a mechanistic understanding of the oocyte polarity defects caused by mutation in the putative Lan receptor Dlar (Poulton, 2006).
Precisely how ectopic DG on the surface of the PFC translates to mislocalizations of posterior polarity markers in the adjacent oocyte remains to be determined, however, several different explanations for this process can be considered. (1) DG down-regulation in the PFC may be necessary to allow the actin-based cortical anchoring of the posterior determinants in the oocyte. (2) The down-regulation of DG after EGFR activation might serve as a cue to the oocyte, which leads directly to MT reorganization and AP axis formation. In this analysis, however, DG overexpression did not result in defects in global microtubule organization or mislocalization of anterior oocyte polarity markers, phenotypes that have been reported in grk and top mutant egg chambers. Furthermore, simply reducing DG levels in non-PFCs by RNAi was not sufficient to mislocalize Stau to nonposterior regions of the oocyte. Therefore, DG down-regulation alone probably cannot serve as the signal to repolarize the microtubule network and, thus, establish oocyte polarity, but it is possible that changes in cell adhesion mediated through the DG/Lan complex could be part of a complex signal involving additional ECM receptors or even other signaling mechanisms that have yet to be identified. A similar model has been proposed for this signal in which changes in cell adhesion between the oocyte and PFCs serve as a nontraditional signal initiating AP axis formation. Alternatively, EGFR-mediated changes in DG/Lan patterns could regulate a novel mechanism that is required specifically for localization of posterior determinants at the oocyte cortex but is independent of the signal provided by the PFC to repolarize the oocyte microtubule cytoskeleton (Poulton, 2006).
(3) The apicobasal defects caused by up-regulation of DG may have led to the loss of apical targeting of the polarizing signal from the PFC, as has been proposed for oocyte polarity defects caused by Merlin mutation. This explanation does not seem likely, however, given the ability of DG RNAi to rescue the CAM phenotype even though the Ras clones still should be unable to produce the signal, because they do not take the PFC fate. Instead, a model is favored in which the apicobasal defects caused by ectopic DG results in apical accumulations of Lan, thereby modifying the ECM between the clones and oocyte so as to preclude diffusion of a secreted signal from the adjacent wild-type cells. Therefore, in the Ras rescue experiment, down-regulation of DG allows the basal restriction of Lan, facilitating diffusion of the polarizing signal from the remaining wild-type cells. The fact that the rescue of the CAM phenotype by DG RNAi in Ras clones was not complete (34% of these egg chambers continued to show some defect in Stau localization) may support this model, because the diffusion of a signal from the neighboring cells probably would not be expected to replace fully the endogenous signal absent from the clone cells in every case. Whether mutations in other genes required for both apicobasal polarity and oocyte polarity also disrupt the ECM will be interesting to discover (Poulton, 2006).
The study of axis formation in the Drosophila oocyte has demonstrated the importance of cell–cell communication in the tightly regulated patterning of the follicle cells, which ultimately leads to the establishment of those axes. The key findings presented here suggest a multifaceted role for EGFR signaling in PFC differentiation and oocyte polarization, highlighting the need for further study of EGFR activity, differentiation of the PFC, and formation of the AP axis (Poulton, 2006).
Cell-matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. This study analysed the role of the secreted serine protease-like protein Scarface in germ-band retraction and dorsal closure -- morphogenetic processes that rely on the activity of integrins and laminins. Evidence that
scarface is regulated by c-Jun amino-terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. It is proposed that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context (Sorrosal, 2010).
During germ band retraction (GBR), the tail end of the germ band, or embryo proper, interacts with the amnioserosa (AS), an epithelium of large flat cells that does not contribute to the larva, and moves to its final posterior position. After GBR, the ectoderm has a gap on its dorsal side that is occupied by the amnioserosa (AS). The dorsal-most cells of the ectoderm, on both sides of the embryo, are in contact with the AS and are called leading edge (LE) cells. During DC, LE cells direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS until they meet and fuse at the dorsal midline. Attention of this study centered on scarf on the basis of its embryonic expression pattern. Two piggyBac insertions, scarf PBss(GFP) and scarf M13.M2(lacZ), were expressed at a high level in LE cells during GBR and DC and at a low level in AS cells. The expression of
scarf was confirmed by in situ hybridization. It was also expressed in the head and tail regions, and in the ventral ectoderm in a segmental pattern (Sorrosal, 2010).
The JNK pathway is activated in LE cells and leads to the expression of the signalling molecule Dpp and the phosphatase Puckered (Puc). The
scarf gene was expressed in the same cells as puc and dpp at the LE. Reduced JNK - Basket (Bsk) in
Drosophila - or expression of a dominant-negative version of Bsk or Puc, which mediates a feedback loop repressing JNK activity, led to the loss of scarf expression in LE cells. Loss of puc activity, which leads to increased levels of JNK activity, or expression of an activated version of JNK -activating kinase (Hemipterous in Drosophila), led to the expansion of the scarf expression domain throughout the lateral ectoderm. Dorsal cells had a stronger response to increased JNK, suggesting that JNK requires the activity of another factor expressed in this region to induce scarf. As stated, the JNK cascade drives the expression of the secreted molecule Dpp. In embryos mutant for the Dpp receptor Thickveins, scarf expression at the LE was not lost and ectopic activation of the Dpp pathway did not induce the ectopic expression of scarf. These results indicate that scarf expression is induced by JNK in LE cells (Sorrosal, 2010).
Homozygosis or trans-heterozygosis for the piggyBac insertions
scarf PBss and scarf M13.M2 caused semi-lethality and survivors had scars on the head, phenotypes that were reverted by precise excision of the transposon elements. As embryos showed no obvious cuticle phenotype, stronger alleles of scarf were generated by imprecise excision of a P element located in the third intron of
scarf (scarf KG05129). One allele (scarf
Δ1.5) was isolated that led to the loss of scarf messenger RNA expression. This is an embryonic lethal allele and
trans-heterozygous combinations of scarf Δ1.5 or Df(2R)nap14, a deficiency that uncovers the scarf locus, with scarf PBss or scarf M13.M2 were semi-lethal and resulted in scars on the head. Homozygous animals for scarf Δ1.5 or Df(2R)nap14 showed similar phenotypes. Their larval cuticles were either dorsally wrinkled or presented a dorsal hole or a U-shaped phenotype, which is suggestive of failures in GBR and DC. A high proportion of embryos showed undifferentiated cuticles. Ubiquitous expression of
scarf largely rescued the scarf Δ1.5 phenotype (Sorrosal, 2010).
Time-lapse recordings indicated that these embryos were delayed in development and showed a failure in GBR. Frequently, the attachment of the AS to the tail end of the germ band was compromised and the germ band did not retract. In embryos that were able to retract, DC started and LE cells began to direct the movement of the epidermal sheets migrating dorsally over the apical
side of the AS. However, either the AS detached from the neighbouring epidermal sheet or the epidermal sheets met and fused at the dorsal midline but the dorsal epidermis reopened. The requirement of scarf in DC was analyzed in greater detail in fixed staged embryos. During the initial steps of DC, lateral ectodermal cells start to elongate dorsally and an actin cable at the LE is formed, which helps to generate a linear fence at the interface between LE and
AS cells. In scarf Δ1.5 embryos, elongation of the lateral ectoderm was compromised frequently, the actin cable was not formed properly, the interface between LE and AS cells became irregular, and eventually rips appeared at the AS-LE interface. The elongation of lateral ectodermal cells and adhesion between AS and LE cells are mediated by the activity of Dpp and JNK activity controls the polymerization of actin into a cable at the LE. In embryos lacking scarf, the activity of the Dpp and JNK signalling pathways was not affected. Thus, Scarf has a role in DC without affecting the activity levels of these pathways (Sorrosal, 2010).
Integrins and laminins have a fundamental role in GBR and DC by mediating interactions between AS cells and neighbouring cell populations. As defects were observed in these processes on scarf depletion and a reduction in scarf levels increased the frequency of cuticle phenotypes caused by depletion of the β-integrin position-specific (βPS) subunit, the contribution of Scarf to the regulation of the expression levels and subcellular localization of these proteins was examined. Cross-sectional views of properly staged wild-type and scarf Δ1.5 embryos stained for βPS integrin revealed similar levels and localization of integrins. The JNK signalling regulates the expression of the βPS integrin subunit during DC. Although ectopic activation of JNK induced increased expression of βPS integrin, ectopic expression of scarf did not exert this effect. The expression of LanA, one of the two existing fly
α-laminins. In wild-type embryos, high levels of LanA were localized at the BM of AS cells. Interestingly, scarf embryos revealed strong defects in the polarized distribution of LanA. It was localized on the apical side of the AS epithelium and its protein levels were largely reduced in the BM. Ubiquitous expression of scarf largely rescued the levels of LanA at the BM. Similar defects were observed in the polarized distribution of LanA in the lateral ectoderm of scarf mutant embryos, and these defects were also rescued largely by the ubiquitous expression of scarf. Apico-basal polarity of AS cells, visualized by the basal localization of βPS integrin and the localization of E-cadherin (E-cad) at the adherens junctions, was not
affected in hypomorphic scarf PBss and scarf
M13.M2 embryos and in some scarf Δ1.5 embryos even though LanA was localized on the apical side. However,
scarf depletion was frequently found to cause defects in the apico-basal polarity of AS cells and this was accompanied by multilayering of the AS epithelium. These observations suggest that before inducing a loss of epithelial integrity, the absence of Scarf caused aberrant localization of LanA on the apical side of the AS epithelium and reduced LanA in the BM, without affecting the distribution of apical and baso-lateral proteins. As integrins are involved in maintaining epithelial polarity, these results suggest that the observed loss of cell polarity is a consequence of reduced integrin-mediated adhesion to the BM. Consistent with this view, defects in the attachment of the AS cells to the
underlying yolk cell were observed frequently (Sorrosal, 2010).
Although the defects observed in scarf Δ1.5 embryos resemble those caused by βPS integrin depletion, only defects in the attachment of the AS with the underlying yolk cells are found in lanA mutant embryos. Mutations in wing blister (wb), the only other fly
α-laminin, cause defects in GBR and in the attachment between AS cells and the posterior germ band. These data suggest that LanA and Wb have a redundant function and that Scarf most probably
promotes BM localization of not only LanA but also Wb. Consistent with the proposed redundancy, halving the dose of
lanA or wb increased the frequency of the βPS integrin loss-of-function cuticle phenotype. The expression of Wb protein in AS cells using the available antibodies and the role of Scarf in the polarized distribution of other BM proteins, such as Perlecan or Collagen IV, were not addressed as these two proteins were not expressed in AS cells (Sorrosal, 2010).
These results indicate that Scarf depletion causes defects in the BM localization of LanA and in epithelial apico-basal polarity. The defects resemble those observed in follicle cells (that is, epithelial cells covering the fly oocyte) mutant for crag, a gene encoding for a protein localized in early and recycling endosomes and proposed to regulate protein transport and membrane deposition of BM proteins. Zygotic removal of
crag induced anomalous localization of LanA on the apical side of AS cells. Zygotic or both maternal and zygotic removal of crag produced cuticles with a weak dorsally wrinkled phenotype, suggesting that Crag has a redundant function with another protein in the fly embryo and that the low BM levels of LanA observed in these embryos are sufficient to exert their function. Interestingly, halving the dose of scarf expression gave rise to a large proportion of crag cuticles with phenotypes similar to those caused by
scarf depletion (Sorrosal, 2010).
Antibodies against Scarf were raised to analyse its subcellular localization. As the antibodies did not work properly at embryonic stages and did not detect endogenous levels of Scarf protein, the wing imaginal disc, a monolayered
epithelium, was used to analyse its subcellular localization. Scarf was not detected in wild-type wing cells. When
scarf and green fluorescent protein (GFP) were expressed in a restricted domain in the wing disc, Scarf was detected not only in the GFP-labelled
scarf-producing cells, but also on the apical side of the epithelium at long distances from the source. When atrial natriuretic factor-GFP (a fusion between the secreted rat atrial natriuretic peptide and GFP) was expressed,
the protein was also observed at long distances from the source; however, it did not accumulate on the apical side of the epithelium. Scarf was found to localize in small punctate structures throughout the cytoplasm of scarf non-producing cells. These structures corresponded either to Rab5-positive early endosomes, to Rab7-positive late endosomes, or to Rab11-positive recycling endosomes. Together, these results indicate that Scarf is secreted apically and is internalized through endocytosis by non-producing cells. To confirm that Scarf is a secreted protein,
Drosophila S2 cells were transfected with either a scarf-myc-tagged transgene or a transgene driving the expression of a myc-tagged membrane-tethered form of Scarf (Scarf-CD2). Scarf was isolated not only from the protein extract of the scarf-myc-expressing cells but also from the supernatant. Transfected Scarf-CD2 and endogenous actin were not observed in the supernatant (Sorrosal, 2010).
This study has characterized the role of scarf, a JNK-regulated gene, in GBR and DC, two morphogenetic processes that rely on cell-matrix interactions between the AS epithelium and neighbouring cell populations. Evidence that Scarf
is a secreted protein expressed in LE cells and involved in promoting the localized accumulation of LanA in the BM of AS cells. Although low levels of Scarf were detected in AS cells, the cuticle phenotype of
scarf mutant embryos and the defects observed in BM localization of LanA and epithelial integrity of AS cells were rescued largely by driving expression of a
scarf transgene only in ectodermal cells, indicating that Scarf is acting as a secreted protein. Three alternative hypotheses might explain the role of Scarf and Crag in the polarized localization of LanA to the BM. As Crag and Scarf are localized specifically on the apical side of the epithelium, they would have a repulsive role, inhibiting the targeting of LanA-containing vesicles with apical membranes. Alternatively, LanA might be secreted apically and basally and be stabilized preferentially on the basal side or degraded on the apical side. As Scarf encodes for a putative serine protease-like protein without catalytic activity, Scarf would function, in this scenario, to facilitate the effect of a cascade of serine proteases involved in the degradation of LanA
in the apical domain of epithelial cells. Finally, in scarf mutant cells, LanA might be transported from the basal to the apical side through transcytosis, a mechanism responsible for the formation of a small apical BM cap over pre-invasive epithelial cells during Drosophila oogenesis (Sorrosal, 2010).
Another secreted serine protease-like protein without catalytic activity, Masquerade, regulates cell-matrix interactions at the somatic-muscle attachment in the fly embryo, a process that also depends on integrin and laminin activity. It is speculated that a number of non-functional serine protease-like proteins, such as Masquerade and Scarf, are regulated temporally and spatially to promote the appropriate localization of BM proteins in a context-dependent manner to
facilitate cell-matrix interactions and to ensure the maintenance of epithelial integrity. Similarly, among the large class of vertebrate functional and non-functional serine protease-like proteins involved in remodelling the extracellular matrix, some of them might exert a similar action (Sorrosal, 2010).
Drosophila integrins have been expressed on the surfaces of cultured
cells and tested for adhesion and spreading on various matrix molecules. PS1
integrin (Multiple edematious wings) is a laminin receptor, PS1 and PS2 (inflated) integrins promote cell spreading on two different Drosophila extracellular matrix molecules, laminin and Tiggrin: PS1 on laminin, and PS2 on Tiggrin. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, lead to a model for the structure of integrin-dependent
attachments in the pupal wings and embryonic muscles of Drosophila. Specifically it is thought that PS2 integrins are restricted to cells of the presumptive ventral layer of the wing and PS1 is restricted to the presumptive dorsal cells. Clones of wing tissue mutant for the PS2 integrin result in blisters on the ventral, but not the dorsal wing surface. It is not known, however, how the opposing cell layers are linked extracellularly. It is proposed that both laminin and Tiggrin are involved in these linkages. Either Tiggrin and laminin bind to one another or bind via other, as yet unidentified ECM molecules (Gotwals, 1994).
A Drosophila UDP-glucose:glycoprotein glucosyltransferase was isolated, cloned and characterized. Its 1548 amino acid sequence begins with a signal peptide, lacks any putative transmembrane domains and terminates in a potential endoplasmic reticulum retrieval signal, HGEL. The soluble, 170 kDa glycoprotein occurs throughout Drosophila embryos, in microsomes of highly secretory Drosophila Kc cells and in small amounts in cell culture media. The isolated enzyme transfers [14C]glucose from UDP-[14C]Glc to
several purified extracellular matrix glycoproteins (laminin, peroxidasin and glutactin) made by these cells, and to bovine thyroglobulin. These proteins must be denatured to accept glucose, which is bound at endoglycosidase H-sensitive sites. The unusual ability to discriminate between malfolded and native glycoproteins is shared by the rat liver homolog. The amino acid sequence presented differs from most glycosyltransferases. There is weak, though significant, similarity with a few bacterial lipopolysaccharide glycotransferases and a yeast protein Kre5p. In contrast, the 56%-68% amino acid identities with partial sequences from genome projects of Caenorhabditis elegans, rice and Arabidopsis suggest widespread homologs of the enzyme. This glucosyltransferase fits previously proposed hypotheses for an endoplasmic reticular sensor of the state of folding of newly made glycoproteins (Parker, 1995).
A novel Drosophila alpha integrin subunit has been identified that associates with betaPS integrin. AlphaPS3 RNA is localized to tissues undergoing invagination, tissue movement and morphogenesis: for example, salivary gland, trachea, midgut, dorsal vessel, midline of the ventral nerve cord, amnioserosa and the amnioproctodeal invagination. AlphaPS3 DNA localizes to the chromosomal vicinity of scab (scb), previously identified by a failure of dorsal closure. Embryos homozygous for the 119 allele of scb have no detectable alphaPS3 RNA. The 1035 allele of scb contains a P element inserted just 5' of the coding region for the shorter of the gene's two transcripts. Mutations in the scb locus exhibit additional defects corresponding to sites of alphaPS3 transcription, including abnormal salivary glands, mislocalization of the pericardial cells and interrupted trachea. Removal of both maternal and zygotic betaPS produces similar defects, indicating that these two integrin subunits associate in vivo and function in the movement and morphogenesis of tissues during development in Drosophila. Phenotypic similarities suggest that laminin A is a potential ligand for this integrin, at least in some tissues (Stark, 1997).
Initial deposition of Laminin is observed between the ectodermal and the mesodermal layers, following germ band extension (stage 11). During germ band retraction, diffuse staining is seen in the mesodermal layer and strongly in cephalic mesoderm. As germ band retraction proceeds, numerous hemocytes react as well as some glial cells. At different focal planes through an embryo one sees hybridizing hemocytes located around the organs where basement membrane deposition occurs, such as the brain, nerve cord, sensory organs and various internal organs, such as the gut (Kusch-Gullberg, 1992). The heart develops from two types of cells: external pericardial cells and internal cardioblasts (which are muscle cells). The dorsal vessel is formed during dorsal closure when the cardioblasts and pericardial cells, both located along the dorsal margin of the epidermis, migrate from both sides of the embryo to meet each other along the dorsal midline. By so doing, they assemble the heart tube. Laminin is deposited along the apical and basal surfaces of the cardioblasts, separating the cardiac and paracardial cell surfaces. Pericardial cells in LanA mutants approach the dorsal midline, but at the dorsal closure stage, the pericardial cells do not form a coherent line as in wild-type heart, but rather dissociate and migrate randomly to different locations within the embryo. Also the cardioblast layer is discontinous in mutant embryos. Laminin appears to be a crucial substrate required to hold pericardial cells and cardioblasts together to enable organogenesis of the dorsal vessel (Yarnitzky, 1995). During morphogenesis of somatic mesoderm at stages 14 to 16, each myotube stretches and extends its leading edge towards its future attachment site in the ectoderm. Having once met the appropriate apodeme, each myotube forms stable contact with this cell. Laminin completely ensheathes the myotube membrane. Despite the normal organization of early somatic mesoderm, careful examination of the pattern of somatic musculature in LanA mutant embryos identifies abnormalities in somatic myotubes. The edges of the ventral oblique muscles never reach their targeted epidermal attachment sites and the morphology of all myotubes is abnormal. Thus Laminin is essential during the somatic myotube extention process (Yartnitzky, 1995). The embryonic midgut develops from both the visceral mesoderm and the endoderm layers. During germ band retraction, anterior and posterior midgut primordia extend toward each other and meet laterally on both sides of the yolk. The movement of the two midgut parts toward each other and the transition of the multilayered mesenchymal cells masses into a highly polarized epithelium are guided by, and dependent upon, the association of the endoderm with the visceral mesoderm. In Lam A mutants, the overall morphogenesis of the midgut and gut occurs as in wild type. However, the initial phase of columnar endoderm polarization is defective, leading to improper arrangement of endoderm along the visceral mesoderm. Thus laminin is essential specifically for the induction of columnar endoderm formation during early midgut development (Yarnitzky, 1995). The role of integrins was examined in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). At the ultrastructural level two types of muscle attachments can be distinguished: direct and indirect. At the direct muscle attachments, single muscles (such as the transverse muscles) attach to epidermal cells directly such that the hemiadherens junctions (HAJs) in opposing cells are separated by only 30-40nm, with a thin line of extracellular electron-dense material in between. These closed paired HAJs are referred to as connecting HAJs. Indirect muscle attachments occur at the segmental border, where the ends of multiple muscles attach at the same epidermal site, and contain extensive extracellular matrix consisting of fuzzy electron dense fibers, separated by up to several micrometers. This is referred to as tendon matrix because, like the vertebrate tendons, it is an extracellular matrix used to attach the muscles. Since HAJs at indirect muscle attachments are not closely paired but connected to the tendon matrix, they are referred to as tendon HAJs. Both types of muscle attachments have a common molecular basis: both contain PS integrins; both are sites were large secreted proteins Tiggrin and Masquerade accumulate; the intracellular appearance of connecting HAJs and tendon HAJs looks similar; connecting HAJs and tendon HAJs can appear together at the same site; they both appear to arise from short connecting HAJs; and both HAJs are separated from the extracellular electron dense matrix by a translucent gap of a few nanometers (Prokop, 1998).
Muscle attachments and neuromuscular junctions were examined ultrastructurally in single or double mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS), and/or their potential extracellular ligand Laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions to extracellular matrix, but not for their intracellular link to the cytoskeleton. The intracellular electron-dense material of connecting HAJs and tendon HAJs connects to microfilaments in the muscles, and to microtubules in the epidermis. The epidermal microtubules are anchored at the other end to apical focal HAJs that connect to the cuticle (Prokop, 1998).
The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. PS2 integrin is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component Laminin A, while adhesion mediated by PS2 integrin does not (Prokop, 1998).
At neuromuscular junctions (NMJs) the formation of functional synapses occurs normally in embryos lacking PS integrins and/or Laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly in embryos lacking laminin A. It is suggested that neuromuscular contact does not require laminin A directly at its point of contact, but requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of Laminin A. In contrast, loss of PS integrin function causes the boutons to make a more extensive contact with the muscle surface. Since no PS integrins are found at neuromuscular contacts it seems likely that the boutons can adhere to more muscle area because the muscle surfaces are more relaxed (allowing them to bend around the bouton) in the severely detached muscles of embryos lacking both PS integrins functions. Adhesion molecules expressed at Drosophila NMJs, like Fasciclin II, Fasciclin III or Connectin, are unlikely to mediate adhesion at the mature embryonic NMJ because they either fade during stage 16 or show no phenotype when mutated. Instead, mutant analysis reveals the existence of yet unknown embryonic adhesion factors downstream of mef2 regulation. Such factors might include laminin receptors that promote adhesion, or other receptors that displace the basement synaptic cell junction. Identification of mef2-dependent receptors might be aided by the use of lamA mutation as a sensitized background (Prokop, 1998).
There are four critical steps in the conversion of a folded single layered wing disc to a flat bilayered wing: apposition, adhesion, expansion and separation. Each step occurs twice, once during the 12 hour prepupal period and again during the 84 hour pupal period. Disc ultrastructure is correlated with the distribution of several elements for each key stage of pupal development: the beta chain of integrin, Laminin A, and filamentous actin. Integrin and Laminin exhibit a mutually exclusive distribution from the adhesion stage onwards. Integrin is present on the basal surface of intervein cells but not on vein cells, whereas Laminin A is absent from the basal surfaces of intervein cells but is present on vein cells. Laminin is not a ligand for integrin in this context. During apposition and adhesion stages, integrin is broadly distributed over the basal and lateral surfaces of intervein cells but subsequently becomes localized to small basal foci. These foci correspond to basal contact zones between transalar processes. The distribution of filamentous actin is dynamic, changing from an apical distribution during hair morphogenesis to a basal distribution as the transalar cytoskeleton develops. Basal adherens-type junctions are first evident during the adhesion stage and become closely associated with the transalar cytoskeleton during the separation stage. Thus, basal junction formation occurs in two discrete steps: intercellular connections are established first and junction/cytoskeletal connections are formed about 20 hours later. These observations provide a basis for future investigations of integrin mediated adhesion in vivo (Fristrom, 1993). The distributions of collagen IV, laminin, and an additional extracellular molecule, the 2G2 antigen (2G2-Ag), were followed immunocytochemically during early wing development. In late third instar larvae, collagen IV and laminin surround the entire wing disc, whereas the 2G2-Ag is limited to the region of the future wing pouch. For the first few hours following eversion of the disc, all three ECM components line the basal surfaces of all epithelial cells in the wing pouch, both those destined to line the wing veins and those destined to become tightly apposed in the large intervein regions. Collagen IV and laminin persist on these cells during the two initial rounds of apposition of dorsal and ventral wing surfaces; later they become restricted to the cells lining the veins. The 2G2-Ag disappears completely quite early in the pupal period. Collagen IV appears to be synthesized at least twice, once in the larva and a second time in the pupa; in between it is enzymatically cleaved and may be eliminated, probably by hemocytes.
In an extreme allele of blistered, the wing is ballooned to form a single internal space. Collagen IV and laminin line all basal wing cell surfaces early in pupal development as they do in the wild type. Later, however, they continue to line the entire cavity of the mutant wing rather than assuming a restricted distribution. In a completely veinless wing (rhomboid/veinlet/vein), collagen IV and laminin are also present generally on basal surfaces at early times, but later are completely absent between the tightly apposed wing layers. The ECM distributions both in wild type wings and in mutants suggest that the matrix plays a role in the establishment of the wing venation pattern. One possibility, strengthened by recent findings regarding ECM receptors in Drosophila, is involvement of these receptors in dorsal-ventral wing layer adhesion. Certain sets of features that distinguish
vein from intervein cells may be linked during cell differentiation and thus help to define these cell phenotypes. The features include cytoskeletal specializations and certain cell surface and ECM molecules (Murray, 1995).
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).
The transmembrane protein Dystroglycan is a highly glycosylated central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Drosophila Dystroglycan (Dg) is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity (Deng, 2003).
Laminin stripes in the basement membrane of
the follicular epithelium are normally organized in the same orientation as the basal actin fibers, suggesting an instructive interaction between the actin cytoskeleton and the ECM through a receptor(s). One explanation for the non-cell-autonomous role of Dg in basal
actin organization is that Dg functions through organizing the Laminin ECM to
affect the basal actin in the neighboring cell. To test this idea further, the orientation of Laminin stripes was examined in wild-type and the
Dg mutant follicle cells. Instead of the orientation perpendicular to
the AP axis seen in the wild type, overall reduction and misorganization of Laminin ECM occurs in the mutant clone and neighboring regions (Deng, 2003).
To test whether Dg is sufficient to organize the Laminin ECM, it was asked
whether overexpression of Dg has any effect on Laminin localization. In stage
10 follicle cells, the majority of the Laminin staining is observed at the
basal side. Noticeably, Laminin is accumulated at the lateral and apical sides
of the follicle cells that overexpress Dg, which is
consistent with the fact that high-level Dg expression is visible at the
apical and basal surfaces of these cells. This result suggests
that Dg can effectively organize the Laminin ECM in Drosophila. The
dotted instead of stripe/line appearance of ectopic Laminin because of Dg
overexpression is consistent with a previous report (Henry, 2001b) that Dg is required for Laminin binding, while Integrin is required for further formation of the
Laminin stripe/line-like structures (Deng, 2003).
Laminin A is necessary in the development of the compound eye. Eyes in LanA mutants have an abnormal shape and a rough appearance at the surface, resulting from changes in the array of hexagonal facets and bristles. There is often a reduction in the number of lens facets leading to a narrowing of the eye. The roughness is associated with the disruption of the normal array of lenses, misplaced and extra bristles, differences in lens shape and occasional fusion of lenses. Defects are seen in leg and wing morphogenesis as well. LanA does not appear to interact with integrin mutants myospheroid or inflated (Henchcliffe, 1993).
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 Scab 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).
Heart development in the Drosophila embryo starts with the specification of cardiac precursors from
the dorsal edge of the mesoderm and continues through signaling from the epidermis. Cardioblasts then become
aligned in a single row of cells that migrate dorsally. After contacting their contralateral counterparts,
cardioblasts undergo a cytoskeletal rearrangement and form a lumen. Its simple architecture and
cellular composition makes the heart a good system to study mesodermal patterning, intergerm layer
signaling, and the function of cell adhesion molecules (CAMs) during morphogenesis. A
focus is placed on three adhesion molecules essential for heart development: faint sausage (fas), shotgun (E-cadherin), and
Laminin A (Lam A). fas encodes an Ig-like CAM and is
required for the correct number of cardioblasts to become specified, as well as proper alignment of
cardioblasts. Those specified in mutants tend to migrate abnormally away from the midline. A similar qualitative phenotype can be achieved by reducing Notch function during cardiac development, suggesting that fas may play a permissive role during Notch/Delta-mediated cell-cell interactions among heart precursors. shg is expressed and required at a later stage than fas; in embryos lacking this
gene, cardioblasts are specified normally and become aligned, but do not form a lumen. Additionally,
cardioblasts of shg mutant embryos show a redistribution of phosphotyrosine as well as a loss of
Armadillo from the membrane, indicating defects in cell polarity. Both Arm and phosphotyrosine are markers for the zonula adherens (ZA). Wild-type cardioblasts have a ZA that mediates the contact between neighboring cardioblasts of the same side of the embryo. The lateral segment of the AZ (between ipsilateral cardioblasts) is unaffected. Cells of opposite sides do not form a junctional complex with each other in shg mutants; they also do not bend into the crescent shape typical of wild-type cardioblasts. The shg phenotype can be
phenocopied by applying EGTA or cytochalasin D, supporting the view that Ca2+-dependent adhesion
and the actin cytoskeleton are instrumental for heart lumen formation. As opposed to cell-cell adhesion,
cell-substrate adhesion mechanisms are not required for heart morphogenesis, but only for maintenance
of the differentiated heart. Embryos lacking the LamA gene initially develop a normal heart, but
show twists and breaks of cardioblasts at late embryonic stages. These findings are viewed in the light of
recent results that elucidate the function of different adhesion systems in vertebrate heart development. The function of Shg during cardioblast lumen formation appears similar to the presumed role of VE-cadherin in vertebrate heart and capillary development. In the chick, VE-adherin expression increases in the endocardial primordium and N-cadherin is turned off (Haag, 1999).
The establishment of the anterior-posterior (AP) axis in Drosophila
requires signaling between the oocyte and surrounding somatic follicle cells
during oogenesis. First, a signal from the oocyte (Gurken) is received by
predetermined terminal follicle cells in which the epidermal growth factor
receptor (EGFR) pathway is activated and a posterior fate is induced. Later, the posterior follicle cells send an unidentified signal back to the
oocyte, that leads to the reorganization of the oocyte's cytoskeletal polarity. This
reorganization is required for proper localization of maternal determinants,
such as Oskar and Bicoid mRNAs, that determine the AP polarity of
the oocyte and the subsequent embryo. When the gene lanA,
which encodes the extracellular matrix component Laminin A, is mutated in
posterior follicle cells, localization of AP determinants is disrupted in the
underlying oocyte. Posterior follicle-cell differentiation and follicle cell
apical-basal polarity are unaffected in the lanA mutant cells, suggesting that
Laminin A is required for correct signaling from the posterior follicle cells
that polarize the oocyte. This is the first evidence that the extracellular
matrix is involved in the establishment of a major body axis (Deng, 2000).
The follicle cell monolayer that encircles each developing
Drosophila oocyte contributes actively to egg development
and patterning, and also represents a model stem cell-derived
epithelium. Mutations in the receptor-like transmembrane tyrosine phosphatase Lar have been identified that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an
indicator of oocyte anterior-posterior polarity. Alterations
in actin filament organization correlate with these defects.
Actin filaments in the basal follicle cell domain normally
become polarized during stage 6 around the anterior-posterior
axis defined by the polar cells (follicle cells lie at the anterior and
posterior poles of ovarian egg chambers beginning at stage 3), but mutations in
Lar frequently disrupt polar cell differentiation and actin
polarization. Lar function is only needed in somatic cells,
and (for Oskar localization) its action is autonomous to
posterior follicle cells. Polarity signals may be laid down by
these cells within the extracellular matrix (ECM), possibly
in the distribution of the candidate Lar ligand Laminin A,
and read out at the time Oskar is localized in a Lar-dependent
manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. Lar acts somatically early in oogenesis, during follicle
formation, and it is postulated that Lar functions in germarium
intercyst cells that are required for polar cell specification
and differentiation. These studies suggest that positional
information can be stored transiently in the ECM. A major
function of Lar may be to transduce such signals (Frydman, 2001).
A clue to the mechanism of Lar action comes from studies on its role in Oskar localization. Posterior follicle cells
must express Lar to ensure that Oskar is localized properly at
the oocyte posterior. When posterior follicle cells lack the ECM component Laminin A (LanA), Oskar localization is usually disrupted. These studies suggest
that LanA and ECM mediate the posterior follicle cell-oocyte
signal. As Lar has been reported to bind to the laminin-nidogen
complex, Lar might act as the LanA
receptor in this pathway. However, it remains less clear how a signal initiated by an interaction between LanA in the ECM and Lar on a posterior
follicle cell would be transduced into the oocyte. Some LanA-containing ECM
resides between the apical surface of the posterior follicle cells
and the oocyte, and it has been proposed LanA interacts directly with the oocyte
surface. An alternative model is proposed. LanA was observed only on the basal side of the follicle cells, and LanA clones induce round eggs. These
observations and the follicle cell autonomous requirement of
Lar for Oskar localization argue that the LanA signal is
received by Lar on the basal surface of the follicle cells and
leads to some change in the receiving cells that is transduced
to the oocyte. This could be via a secondary signal, or by
changes in the structural or adhesive properties of the cells that
can locally affect the oocyte surface with which they come into
contact. Lar mutation does not affect the apical basal polarity of
follicle cells, because the apical-basal asymmetry of actin staining is maintained and multiple-layered follicle cells are never observed (Frydman, 2001).
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).
Tiggrin is a secreted glycoprotein that contains an RGD motif and is
considered to be a ligand of the PS2 integrin. Embryos homozygous for a loss of function allele of Tiggrin have a subtle Fas II phenotype reminiscent of integrin
mutants. CNS axon tracts are wavy, and no midline axon guidance errors
are seen. Labeling of the most lateral axon tract is interrupted
between segments. Like the integrin genes, tig also has a semidominant
interaction with slit. Fas II labeling of fascicles between segments is reduced. Midline guidance errors are seen in one in three segments (Stevens, 2002).
Drosophila Laminin is a trimer of three proteins: Laminin A,
B1, and B2. Laminin is known to be a ligand
of PS1 integrin and possibly other integrins as well. Mutants have not been isolated for the B1 and B2 chains; however, a loss of function allele for lanA encoding the A chain has been characterized. The Fas II
phenotype of the lanA mutant is nearly wild type, revealing
midline guidance errors in 4% of segments. When doubly heterozygous with
sli, in sli/+;lanA/+ embryos, the
frequency of midline crossovers is >30% (Stevens, 2002).
Does a change in lanA function also affect integrin function
in CNS axon tract formation? lanA
interaction with scb was examined because Laminin is not known to be a
ligand of alphaPS3/4 (encoded by scb), and scb has
a strong semidominant interaction with slit. Both
lanA and scb reveal midline guidance errors when homozygous. However, in the scb/+;lanA/+ double heterozygote, midline
guidance errors are not seen. Nevertheless,
this genotype shares aspects of the integrin CNS phenotype:
defasciculation and interruptions in Fas II labeling of the most
lateral fascicle. This suggests function of both genes in a common or parallel pathway. If the interaction of scb and lanA is independent of the
interaction of either gene with sli, then the phenotype of
the triple heterozygote scb/sli;lanA/+ would reflect the
addition of the scb/sli, scb/+;lanA/+,
and sli/+;lanA/+ phenotypes. The degree of
defasciculation and midline guidance errors in all axon tracts of the
triple heterozygote appears to be additive.
However, a narrowing of the CNS and the medial displacement of all axon
tracts are also seen in the triple heterozygote. This phenotype is
typical of mutants in genes required for midline guidance and is
not a component of the integrin mutant phenotype. The synergistic
interaction of these three genes suggests dosage-dependent function
for each gene in common or parallel pathways (Stevens, 2002).
Thus, a reduced level of expression of
the genes for four integrins (alphaPS1, alphaPS2, alphaPS3/4, and ßPS1) or
two integrin ligands (Tiggrin and Laminin) increases the probability that CNS axons make pathfinding errors when slit expression
is reduced. Expression of the integrins Tiggrin and Laminin A has been
demonstrated in the CNS. Integrin
expression is not localized and may be expressed in both glia and
neurons. Overexpression of alphaPS3 or Laminin A in motoneurons affects
axon guidance. Loss of function of the integrins
disrupts axon fasciculation and longitudinal axon fascicle placement in
the embryonic nerve cord but does not clearly affect axon guidance. These observations have been extended in this study,
with different alleles of the integrins, demonstrating a similar
function for alphaPS3/4, and revealing axon fascicle phenotypes for loss
of function of integrin ligands Tiggrin and Laminin A. The mutant
phenotypes share common elements: mild phenotypes show wavy axon tracts
and reduced Fas II labeling between segments, whereas severe phenotypes
include defasciculation and fascicle displacement, including midline
axon guidance errors. The integrins have different extracellular
ligands. Therefore, the integrins contribute similarly to axon tract
integrity, independent of the ligand that they bind (Stevens, 2002).
Integrin phenotypes in the CNS do not demonstrate a direct role for
integrins in growth cone guidance. In contrast, perturbation of midline
growth cone repellent signals results in a medial narrowing of the CNS
and ectopic midline crossing of longitudinally projecting axons, rather
than defasciculation and displacement of axon tracts. One feature of
integrin and tiggrin phenotypes shared with robo and dock mutant phenotypes is a thinning or loss of Fas II
labeling in the most lateral axon fascicle. This fascicle expresses Fas II late in embryogenesis. This phenotype may reflect impaired or
delayed development of independent fascicles in the nerve cord, as
implicated by studies of robo function (Stevens, 2002).
The semidominant interaction of all integrins, Tiggrin, and Laminin A
with slit is more prevalent than might be expected if the
integrins play a specialized role in Slit signaling. scab also has a dramatic semidominant interaction with dock
(Nck), which functions in diverse axon guidance events. scb/dock double heterozygotes have disrupted longitudinal, commissural, and peripheral axon tracts. A similar genetic test suggests that ßPS integrin modulates RhoA activity and axon stability in the mushroom body. These diverse
phenotypes reflect an adhesive function of the integrins that reduces
the responsiveness of growth cones to guidance signals. Independent evidence suggests that this occurs in the growth cone, but a role for
integrin in the glia that emit guidance signals cannot be discounted (Stevens, 2002).
The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood. This study established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane. Genes for the two collagen IV subunits (see viking and Collagen type IV) and the four laminin subunits were nocked down individually via UAS-RNAi using ubiquitous and tissue-specific GAL4 drivers.
The basement membrane was found to endow otherwise susceptible target tissues with self-tolerance that prevents autoimmunity, and it was further demonstrated that laminin is a key component for both structural maintenance and the self-tolerance checkpoint function of the basement membrane. Moreover, cell integrity, as determined by cell-cell interaction and apicobasal polarity, was found to function as a second discrete checkpoint. Target tissues became vulnerable to blood cell encapsulation and subsequent melanization only after loss of both the basement membrane and cell integrity (Kim, 2014)
Laminin A:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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