shotgun
Maternal SHG mRNA is distributed uniformly in early embryos. During cellularization, SHG zygotic transcript is found in all blastoderm cells, but is excluded from cells of the presumptive mesoderm and endoderm that will convert into mesenchymal cells after gastrulation. Later in development, two mesodermally derived epithetial cell sheets express shg: the dorsal vessel and the gonadal sheet. The endoderm initiates zygotic shg expression just before its cells convert back into epithelial cells forming the lining of the larval midgut.
The amnioserosa [Images], the ectodermal epithelia and its epithelial derivatives (epidermis, tracheal system, foregut, hindgut, Malpighian tubules, and salivary glands) express shg continuously. shg is downregulated in neural precursors while they undergo an epithelial-mesenchymal transition and thereby segregate from the neurectodermal epithelium. shg is active however, in midline cells and the subperineurial glial sheath that forms the blood-brain barrier (Tepass, 1996).
Cadherin-N mRNA is first seen within nuclei of presumptive mesodermal cells prior to gastrulation at stage 5. mRNA transport to the cytoplasm starts at about stage 6-7, and the messengers are distributed throughout the cytoplasm by stage 8. Cadherin-N protein first appears at intercellular contacts in the mesoderm at stage 9, and then the protein is detected at boundaries of mesodermally derived cells that inititate transcription of the Shotgun gene at stage 13. Cadherin-N also appears in developing neural cells, presumably at their postmitotic stage; subsequently, Cadherin-N accumulates in axons of the entire CNS. At the subcellular level, neuronal processes including growth cones are labeled. Gastrulation and neurulation coincide with a switch of cadherin expression from Shotgut to Cadherin-N. Glial cells do not express Cadherin-N (Iwai, 1997).
During Drosophila gastrulation, morphogenesis occurs as a series of cell shape changes and cell
movements that probably involve adhesive interactions between cells. The dynamic aspects of cadherin-based cell-cell adhesion were examined in the morphogenetic events to
assess the contribution of such activity to morphogenesis. Shotgun and Cadherin-N show complementary expression
patterns in the presumptive ectoderm and mesoderm at the mRNA level. Switching of
cadherin expression from the Shotgun to the CadN type in the mesodermal germ layer occurs downstream
of the mesoderm-determination genes twist and snail. In contrast to twi and sna mutations, folded gastrulation mutants show normal replacement of Shotgun with CadN in cells corresponding to the mesoderm. Shotgun mRNA is present uniformly in the embryo until late stage 5, but it begins to disappear in the presumptive mesoderm shortly before the onset of ventral furrow formation. After stage 7 Cadherin-N mRNA is visible in the mesoderm. However, examination of cadherin protein expression
patterns shows that considerable amounts of Shotgun remains on the surfaces of mesodermal
cells during invagination, while CadN does not appear on the cell surfaces at this stage. Further
immunocytochemical analysis of the localizations of Shotgun and its associated proteins Armadillo
(beta-catenin) and Dalpha-catenin reveals dynamic changes in their distributions that are
accompanied by changes in cell morphology in the neuroectoderm and mesoderm. Shotgun, together with Armadillo and Dalpha-catenin, most strongly accumulate at apical contacts of neuroectodermal cells, at the same time that large apical junctions (AJs) are observed at the corresponding sites. As soon as the germ band starts to elongate (stage 8), the apical accumulation along lateral cell surfaces becomes disordered or obscure. Adherens junctions, based on the cadherin-catenin system, change their location,
size, and morphology. At this time large AJs are rarely found. During mesodermal invagination, as invaginating mesodermal cells are converted from wedge-shaped to round cells, Shotgun is gradually redistributed from AJs to a uniform distribution over the entire cell surface, including the cell contact-free areas in rounded mesodermal cells at stage 8. After this stage, Shotgun is completely eliminated from the mesoderm, and Arm and Dalpha-catenin are reduced to undetectable levels. These dynamic aspects of cadherin-based cell-cell adhesion appear to be
associated with the following: (1) initial establishment of the blastoderm epithelium; (2) acquisition of
cell motility in the neuroectoderm; (3) cell sheet folding, and (4) epithelial to mesenchymal conversion
of the mesoderm. These observations suggest that the behavior of the Shotgun-catenin adhesion
system may be regulated in a stepwise manner during gastrulation to perform successive
cell-morphology conversions. Also discussed are the processes responsible for loss of epithelial cell polarity and
elimination of preexisting Shotgun-based epithelial junctions during early mesodermal
morphogenesis (Oda, 1998a).
Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short stop (shot). In these fusion cells, the apical surface determinant Discs lost (Dlt: now redefined as Drosophila Patj) is subsequently deposited and new lumen forms along the track. In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led to an investigation of whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. It is therefore proposed that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization (Lee, 2002).
The tracheal lumen is initially closed at branch tips. Concurrent with branching morphogenesis, specialized cells at branch tips, known as fusion cells, join branches into a continuous tubular network. This process of anastomosis requires each fusion cell to recognize its partner in the adjacent hemisegment and to form a lumen that connects the two branches. Shotgun, the Drosophila homolog of the cell adhesion molecule E-cadherin is integral to the initial fusion cell contact. Mutations in shotgun affect tracheal branch extension and lumen formation at anastomosis sites, as do mutations in armadillo, the Drosophila homolog of its effector ß-catenin. E-cadherin and ß-catenin control cell polarity and tube extension in culture, suggesting an evolutionarily conserved role for cadherin-mediated cell adhesion in apical surface regulation (Lee, 2002).
The results presented here provide further insights into how the cytoskeleton and associated proteins support contact formation and subsequent apical surface remodeling. The F-actin- and microtubule-binding domains of Shot are required to maintain and remodel E-cadherin contacts and to assemble a track of F-actin and Shot in fusion cells. This track initiates at the E-cadherin contact and extends outwards from it to connect with the existing apical assemblies of F-actin and Shot. It is proposed that the track guides new apical surface formation. Apical surface determinants and membrane appear to accumulate along the track, possibly by spreading from existing apical concentrations. This track may also enable the fusion cells to contract and to draw the existing lumenal surfaces closer, as fusion cells appear notably less compact in shot mutant embryos (Lee, 2002).
shot is required in neurons for growth cone motility. shot is also required to remodel E-cadherin-containing contacts between tracheal fusion cells. Surprisingly, Shot proteins perform these distinct morphogenetic roles using different combinations of the same cytoskeletal interaction domains. In fusion cells, the binding sites for F-actin and microtubules appear functionally redundant. The F-actin binding domain is essential when the GAS2 microtubule binding site is absent, and the GAS2 microtubule binding site is essential when the F-actin binding site is absent. By contrast, during axon extension, the Shot behaves as an F-actin/microtubule cross-linker because the cytoskeletal interaction domains are both individually essential and required in the same molecule (Lee, 2002).
These observations suggest that direct interactions between Shot and cytoskeletal proteins organize the cytoskeleton in fusion cells. The F-actin and microtubule domains may directly enable the accumulation of their cytoskeletal partners at the E-cadherin contact. In support of this hypothesis, the structurally similar F-actin binding domain of plectin alters F-actin organization and the GAS2 motif stabilizes associated microtubules against depolymerization in cultured cells. Since Shot’s interactions either with F-actin or with microtubules suffice to organize both cytoskeletal elements, binding to either F-actin or microtubules may then enhance other organizing interactions between F-actin and microtubules (Lee, 2002).
These other interactions may involve molecules required for E-cadherin signaling. E-cadherins are physically linked to F-actin via the ß-catenin/alpha-catenin complex and to dynein, a microtubule-based motor, via ß-catenin. They can further regulate actin dynamics via association with p120, a RhoA antagonist; E-cadherins also stabilize microtubule minus ends in cultured cells. E-cadherin signaling may therefore affect other proteins mediating interactions between F-actin and microtubules. Candidates include other F-actin/microtubule cross-linkers, regulators of Rho family GTPases that bind to microtubules and F-actin-based motors that form complexes with microtubule-based motors. Further analysis will be necessary to identify these other molecules in fusion cells: these other cytoskeletal regulators may permit residual anastomoses in shot mutant embryos (Lee, 2002).
In cells throughout the trachea, reduced RhoA activity disrupts lumen formation and partially disrupts Dlt (now Patj) localization. Tracheal expression of RhoAN19 does not appreciably affect E-cadherin localization. In cultured epithelial cells, E-cadherin localization is also resistant to RhoAN19. These findings are consistent with RhoA functioning downstream of or parallel to E-cadherin. E-cadherin-associated p120ctn negatively regulates RhoA, but whether a similar pathway operates in Drosophila is unknown (Lee, 2002).
In fusion cells, RhoA can also function upstream of E-cadherin, as constitutively active RhoAV14 affects E-cadherin localization selectively in these cells. E-cadherin distribution is more dynamic in fusion cells than in other tracheal cells, and may therefore be more sensitive to RhoAV14. RhoAV14 also affects new E-cadherin contacts in culture. Further experiments will reveal whether Shot, RhoA and E-cadherin function in a common, evolutionarily conserved pathway to regulate apical surface remodeling in fusion cells (Lee, 2002).
Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. The tracheal system of Drosophila was used to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. A reduced Rac activity leads to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibits dynamic epithelial cell rearrangement. In contrast, hyperactivation of Rac inhibits assembly of newly synthesized E-Cadherin into cell junctions and causes loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules (Chihara, 2003).
Cadherin-based cell adhesions are vital to maintain the morphological and
functional features of the epithelium of multicellular organisms. During
morphogenesis of the epithelia, cell adhesions must be disrupted and
re-assembled in a regulated manner to allow movement of individual cells in
the epithelia. In vivo analyses have demonstrated that a reduction in Rac
activity prevents cell rearrangement. This phenotype is associated with an
increase in the level of E-Cadherin and its associated molecules, and
expansion of E-Cadherin localization to the basolateral membrane. It is inferred that increased E-Cadherin expression consolidates cell adhesiveness.
Hyperactivation of Rac prevents incorporation of newly synthesized E-Cadherin
into cell junctions and reduces cell adhesiveness, transforming the tracheal
epithelium into mesenchyme. It is suggested that switching of Rac between active
and inactive states promotes turnover of the complex containing E-Cadherin at
the cell junction, and maintains the plasticity of the tracheal epithelium to
allow branching morphogenesis (Chihara, 2003).
Expression of a dominant-negative form of Rac 1
greatly reduces cell rearrangement required for partitioning cells into the
stalk of the dorsal branch. Overproduction of this form, Rac 1N17, would shift the
cellular pool of Rac toward the inactive GDP-bound state. It is suggested that
turnover of E-Cadherin at a proper level requires a high level of Rac
activity. However, since highly active movement of cell extensions in the cells at
the tip is still visible, it is suggested that the ability of those cells to move
toward their target is mostly intact. A stronger reduction in Rac activity
might be required to demonstrate a role for Rac in promoting cell extensions,
as proposed from studies on tissue culture cells (Chihara, 2003).
Time-lapse analysis demonstrates that reduced Rac activity inhibits
cell rearrangement during branching of tracheal tubules. Under this condition,
the amounts of cadherins and catenins were increased and filled the cell
membrane. This phenotype is different from the phenotype of E-Cadherin-GFP
overexpression, which does not inhibit cell rearrangement. It is
suggested that a reduction in Rac promotes the association of cadherin-catenin
complexes with the cell membrane and stabilization of these complexes.
Activation of Rac resulta in an opposite phenotype characterized by the loss
of E-Cadherin and cell dissociation, and in prevention of E-Cadherin-GFP from
accumulating at apical cell junctions. All of these observations are
consistent with a hypothesis that Rac regulates the formation of
cadherin-catenin complexes at the cell junction. Incorporation of a
cadherin-catenin complex into the cellular junction would explain
stabilization of the complex when Rac activity is reduced. Possible modes of
Rac action on cadherin include apical transport and assembly/stabilization of
the complex. It is suggested that the inhibitory action on the cadherin cell
adhesion system is a general property of Rac in the Drosophila
embryo (Chihara, 2003).
How E-cadherin controls the elaboration of adherens junction-associated cytoskeletal structures crucial for assembling tubular networks was investigated. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Short stop (Shot), a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to ß-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct, evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly (Lee, 2003).
Junctional contacts between cells are important for organizing the
cytoskeleton and regulating cell polarity. The large size
of plakins and their modular abilities to bind different cytoskeletal elements
make them potentially well suited to play key organizational roles.
However, except in the case of desmosomes, where the plakin desmoplakin
appears to be a crucial for organizing junction-associated cytoskeleton,
functional association of plakins with other cell-cell junctions has not been
described (Lee, 2003).
In selected cell types, Shot localizes with proteins of
the adherens junction and may play a role in adherens junction-mediated
organization of the cytoskeleton. It is proposed that Shot and E-cadherin form a
feedback loop in which E-cadherin, via ß-catenin, recruits Shot to new
contacts between the fusion cells and Shot stabilizes the contacts. The
cytoskeleton organizes around these contacts because adherens junction
associated Shot promotes the assembly of an F-actin/microtubule-rich track.
This track grows to span the fusion cells, extending the reach of the
junctions through the cells. The recruitment mechanism may be indirect in that
new adherens junctions in fusion cells are centers for cytoskeletal assembly,
and Short Stop binds F-actin and microtubules. Alternatively, Shot may
associate directly with E-cadherin or associated proteins. The assembly of
Shot with F-actin and microtubules may stabilize E-cadherin contacts simply by
bringing in cytoskeletal proteins that bind E-cadherin or associated proteins.
For example, EB1, which is present in the fusion track, co-immunoprecipitates
with a C-terminal fragment of Shot in cultured cells and associates with APC. APC interacts with ß-catenin to control tubulogenesis in vitro (Lee, 2003).
It is proposed that the assembly and maturation of a cytoskeletal intermediate
are two E-cadherin-dependent steps in tracheal cell fusion. Imaging of fixed and
live embryos suggests that fusion proceeds through the assembly and maturation
of a cytoskeletal track associated with adherens junctions. The track forms
after contact between the fusion cells, and persists for ~1 hour before
fusion occurs (Lee, 2003).
In this model, the ß-catenin-binding site and the juxtamembrane site
in the E-cadherin cytoplasmic domain operate sequentially and in the same
E-cadherin molecule to promote fusion. In mutant embryos in which either
ß-catenin or its binding site is defective, fusion cells make contact but
track assembly is not observed. These data suggest that E-cadherin may
initiate track assembly via ß-catenin. A mutation in the juxtamembrane
site dominantly inhibits track maturation. Microtubules are generally absent
from fusion tracks in these embryos, though some F-actin and Shot assembly
occurs. In E-cadherin/shotgun (shg) mutant embryos,
E-cadherin bearing this juxtamembrane mutation supports a low level of
F-actin/Shot track formation, but the tracks do not mature. In addition, this
juxtamembrane mutant E-cadherin causes progressive delocalization of the
apical tracheal cytoskeleton in shg mutant embryos (Lee, 2003).
Both the ß-catenin and juxtamembrane binding sites are required for
E-cadherin localization to adherens junctions, although only the juxtamembrane
mutation seems to interfere with endogeneous E-cadherin localization. The
results suggest that like mammalian E-cadherin, an evolutionarily conserved
juxtamembrane site is required for some E-cadherin functions. Similar effects
of mutations in the juxtamembrane site were observed in mammalian tissue
culture cells. However, juxtamembrane site function in Drosophila
E-cadherin probably does not require p120 (Lee, 2003).
Dominant effects on localization appear sensitive to expression levels,
whereas effects on fusion are less so, suggesting that defects in localization
are not enough to explain the defects in track maturation. Possibly, effects
on localization also reflect defects in organizing the cytoskeleton, as has
been observed in studies in which dominant alleles of Rho family GTPases
affect cadherin localization in culture (Lee, 2003).
It is proposed that the ß-catenin-binding site and ß-catenin are
required for track assembly, and that the juxtamembrane site regulates other
proteins involved in a later maturation step. This later step
likely requires microtubules. The microtubules or associated proteins may
reinforce the initial F-actin assembly in the track, as F-actin in fusion
tracks appears to be abnormally or poorly assembled in embryos expressing
AAA-JXT mutant E-cadherin in tracheal cells. The microtubules appear to be also
required for remodeling the fusion cell apical surfaces and also for bringing
them together to fuse. In embryos expressing AAA-JXT mutant E-cadherin in
tracheal cells, fusion cell apical surfaces do not develop or seal gaps at
appropriate times, and fusion tracks persist substantially longer, if they
resolve at all (Lee, 2003).
The microtubule regulated steps during fusion therefore likely involve
effects on F-actin dynamics. Microtubule-associated factors that may regulate
the F-actin cytoskeleton include Rac GTPase and exchange factors for Rho GTPase. Rac1 affects E-cadherin dependent adhesion in tracheal cells and a mutation in the juxtamembrane site in mammalian E-cadherin analogous to the one described in this study affects Rac activation. RhoA activation inhibits fusion track assembly.
Downstream interactions between F-actin and microtubules, such as those
mediated by Shot, may vary with cell type to produce distinct morphogenetic
outcomes. Further studies of tracheal tube fusion, a genetic system in which
adherens junction associated structures can be visualized in living embryos,
promises to identify the regulatory molecules that allow E-cadherin to direct
F-actin and microtubule assembly from the ß-catenin binding and
juxtamembrane domains (Lee, 2003).
Embryo formation requires tight regulation and coordination of adhesion in multiple cell types. By imaging, 3D reconstructions and genetic analysis during posterior midgut morphogenesis in Drosophila, a novel requirement was found for the conserved FGF signaling pathway in maintenance of epithelial cell adhesion, by modulation of zygotic E-cadherin. During Drosophila gastrulation, primordial germ cells (PGC) are transported with the posterior midgut while it undergoes dynamic cell shape changes. In Branchless and Breathless mutant embryos zygotic E-cadherin is not targeted to AJs causing midgut pocket collapse impacting on PGC movement. The ventral midline also requires FGF signaling to maintain cell-cell adhesion. FGF signaling regulates the distribution of zygotic E-cadherin during early embryonic development to maintain cell-cell adhesion in the posterior midgut and the ventral midline, a role that is likely crucial in other tissues undergoing active cell shape changes with higher adhesive needs (Pares, 2015).
The propagation of force in epithelial tissues requires that the contractile cytoskeletal machinery be stably connected between cells through E-cadherin-containing adherens junctions. In many epithelial tissues, the cells' contractile network is positioned at a distance from the junction. However, the mechanism or mechanisms that connect the contractile networks to the adherens junctions, and thus mechanically connect neighboring cells, are poorly understood. This study identified the role for F-actin turnover in regulating the contractile cytoskeletal network's attachment to adherens junctions. Perturbing F-actin turnover via gene depletion or acute drug treatments that slow F-actin turnover destabilized the attachment between the contractile actomyosin network and adherens junctions. This work identifies a critical role for F-actin turnover in connecting actomyosin to intercellular junctions, defining a dynamic process required for the stability of force balance across intercellular contacts in tissues (Jodoin, 2015).
Connection of tubules into larger networks is the key process for the development of circulatory systems. In Drosophila development, tip cells of the tracheal system lead the migration of each branch and connect tubules by adhering to each other and simultaneously changing into a torus-shape. This study shows that as adhesion sites form between fusion cells (FCs), myosin and microtubules form polarized bundles that connect the new adhesion site to the cells' microtubule-organizing centres, and that E-cadherin and retrograde recycling endosomes are preferentially deposited at the new adhesion site. It was demonstrated that microtubules help balancing tip cell contraction, which is driven by myosin, and is required for adhesion and tube fusion. Also, retrograde recycling and directed secretion of a specific matrix protein into the fusion-cell interface promote fusion. The study proposes that microtubule bundles connecting these cell-cell interfaces coordinate cell contractility and apical secretion to facilitate tube fusion (Kato, 2016).
Previous studies showed that F-actin-enriched cell protrusions form in the tip of migrating tracheal branches. This study showed that tracheal FCs form polarized microtubule bundles oriented towards the leading edge of the migrating cells. The function of these microtubules is twofold: to concentrate E-cadherin to the newly contacted cell interface and to initiate the formation of new adherens junctions. The E-cadherin that accumulated at the new cell interface is not recycled from the cell surface, but is instead drawn from a newly synthesized pool and recruited preferentially to the FC contact site, and not to existing adherens junctions between FCs and stalk cells. It is speculated that the forward reorientation of the MTOC and the polarization of the microtubule plus ends towards the leading edge underlie the preferential deposition of E-cadherin at the FC contact site. One possible mechanism of preferential deposition is considered, in which microtubules transport endosomes containing E-cadherin towards the contact site, and attempts were made to test this model by imaging vesicular trafficking of the complex containing E-cadherin-GFP and other adherens junction components. No definitive evidence for this model was found. However, the Golgi apparatus was found to shift forward, towards the FC contact site, and that an E-cadherin-GFP signal increased in the plasma membrane before becoming concentrated at the contact site. Based on these observations, a model is favored in which the relocalization of the Golgi apparatus near the leading edge of the FC provides a source for E-cadherin that is deposited locally in the plasma membrane and the trans association of E-cadherin between the FCs nucleates a further concentration of E-cadherin via cis clustering. MTOC components have been shown to be located apically in stalk cells and the microtubule function is required for the apical assembly of adherens junction proteins Par-3 and E-cadherin through regulation of recycling endosomes. This mechanism appears different from FCs, as assembly of new adherens junction occurs in the cell interface enriched with microtubule plus ends opposite to the centriole located in the proximal side (Kato, 2016).
A second microtubule function was discovered in this study, which was to equalize the contraction in FC pairs after contact. The coordinated contraction in FC pairs pulls two FC-stalk cell junctions simultaneously towards the FC contact site. The contractile force comes from a myosin-driven process; the microtubules may serve as a 'ratchet' to fix the length of the FCs after each round of contraction. When microtubules were inhibited, the FCs relaxed to their original length after contracting, which delayed the overall fusion process. When microtubules were destabilized, branch fusion proceeded, albeit with delays and imbalances, and fusion was eventually completed. Even in this condition, the conversion of the FC cells into a torus shape occurred simultaneously, suggesting that there is a mechanism to coordinate the fusion event in FC pairs. The proposed ratchet-like function of microtubule must in some way be linked to the contractile activity of myosin. One good candidate molecule for coupling the actomyosin contraction to the microtubule function is Short-stop (Shot), which belongs to the conserved spectraplakin family of cytoskeletal proteins, and was shown to be required for tracheal branch fusion. The involvement of microtubules and Shot in the ratchet-like mechanisms observed in several contraction-dependent morphogenetic events should be tested in the future (Kato, 2016).
It is interesting to note that balancing of the force applied to the E-cadherin conjugated cell interface via microtubule plus ends is similar in configuration to the mitotic spindle, where microtubule plus ends attached to the kinetochore of each of paired sister chromosomes applies pulling force to each spindle pole. The equal number of cadherin-catenin complex in each side of the FC interface associated with trans-conjugated E-cadherin pairs in the FC interface may provide a platform for microtubule plus end attachment for generating balanced contractile force (Kato, 2016).
When FCs are fully contracted, the plasma membrane of the two adherens junctions in each FC are connected in a single burst and cell pairs are converted simultaneously into a torus shape. This process requires ADP-ribosylation factor-like 3 (Arl3) GTPase, which associates with the microtubules and intracellular vesicles concentrated at the FC contact site. This study showed that Arl3 is required for directed Serp trafficking, and that GFP-Rab9 overexpression overrides the requirement for Arl3. It is proposed that the microtubule-dependent transport of the Golgi apparatus and endosomes facilitates the concentration of Rab9 and Arl3 at the FC contact site, where they act together to increase the concentration of Serp in the lumen. The deacetylation of chitin converts it to the more hydrophilic form chitosan. The increase in water absorption by chitosan would cause the luminal matrix gel to swell, simultaneously pushing the plasma membranes of the FC interface closer to the plasma membrane of the FC-stalk cell interface so that the membrane-fusion machinery triggers the conversion of the paired FCs into a torus shape (Kato, 2016).
A number of issues remain to be explained. Lumen formation in FCs was clearly detected in arl3 mutants, but not in the normal context. This is probably because very small lumen is sufficient to trigger fusion of wild-type FCs. Although Arl3 is absolutely required for fusion, Rab9 and Serp are not, suggesting that the proposed luminal-matrix swelling due to chitin deacetylation is not the sole mechanism of plasma membrane fusion, and additional Arl3-regulated process of fusion control must exist. Moreover, additional Rab9 cargo that acts together with Serp to rescue the arl3 mutants is predicted. To uncover the entire fusion process, it will be necessary to search for additional Arl3 and Rab9 targets, and to analyse FC-specific membrane trafficking and secretion (Kato, 2016).
Although Snail is essential for disassembly of adherens junctions during epithelial-mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, the behavior of E-cadherin-rich junction clusters were tracked, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. These findings provide in vivo evidence for mechanosensitivity of cell-cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT (Weng, 2016).
This study shows that during Drosophila gastrulation, subapical junctions are repositioned toward the apical surface and are strengthened as the cortical tension increases. Both these phenomena follow apical myosin activation and thus may reflect a mechanosensitive response of junctional complexes to the tension generated by this activation of myosin. The junctional responses occur on the time scale of individual myosin pulses and are temporally correlated with those pulses. Such junctional changes depend on myosin activity but do not require Sna, given that ectopic myosin activation recapitulates similar junctional responses in Sna-negative tissues. This phenomenon may not be restricted to Drosophila embryos. The increased contractile actomyosin on the apical cortex of human cell lines deficient for the cortex actin regulator Merlin is associated with a condensation of adherens junctions toward the apical surface, suggesting that the response of adherens junctions to cortical tension can be of general significance (Weng, 2016).
The changes in junction mass and density suggest that, rather than being simple passive anchors for contractile actomyosin filaments, adherens junctions respond to the contractile actomyosin by restructuring and repositioning themselves, potentially involving aggregation and rearrangement of E-Cad molecules within the plasma membrane or vesicle-based redistribution of E-Cad. Indeed, actomyosin organization has been shown to be critical in the lateral clustering of E-Cad molecules. The change in E-Cad clustering is considered an active mechanosensitive mechanism to strengthen the adhesion. Alternatively, the adhesion can also be remodeled through the vesicle-based mechanisms, and endocytosis of E-Cad has been shown to be up-regulated when junctions are under actomyosin-generated stress. The repositioning could also arise through restructuring rather than passive dragging, if for example recycling and turnover rates in the basal regions of the junctions differ from apical regions. Overall, regardless of the underlying mechanism, this mechanosensitivity may be advantageous, providing a direct self-corrective mechanism that allows junctions to adjust their localization and intensity to match the mechanical force they experience (Weng, 2016).
Although the molecular mechanism for the junction strengthening requires further investigation, the data suggest that it is resistant to the posttranscriptional disassembly of adherens junctions downstream of Sna. The phenotype of myosin knockdown in this study resembles that previously described for cta; T48 double mutants, in which apical actomyosin cannot be activated and junctions are lost only in the ventral mesodermal cells. In all scenarios in which Sna expression is associated with junction loss (ventral cells in cta; T48 mutants, ventral cells in myosin knockdown mutants, and ectodermal cells with ectopic Sna expression), Sna is expressed in cells in the absence of myosin contractility. Maintenance of adherens junctions ultimately relies on the balance between assembly and disassembly rates of junctional components. Thus mechanical force likely modulates the assembly/disassembly balance and therefore remains in a homeostatic relationship with the junctions bearing the force (Weng, 2016).
In the early stages of embryogenesis analyzed in this study, E-Cad is maternally provided and thus not subject to direct transcriptional repression. The disassembly of junctions in the absence of myosin contraction must therefore reflect a posttranscriptional regulation on junctions, likely performed by one or several of Sna’s transcriptional targets. Much effort has been invested in identifying transcription targets of Sna, but it is not known which, if any, of its known targets might play such a role. One mesodermally expressed gene, Traf4, is required for fine-tuning junction morphology, but its expression appears to depend on the other mesodermal determinant, Twist, rather than Sna. One gene repressed by Sna in Drosophila mesoderm, bearded, is required for the subapical positioning of adherens junctions in cells not expressing Snail. It is not clear, however, whether Bearded plays a direct role in junction disassembly or a more general role in apical polarity or the apical myosin contractility that drives repositioning. The posttranscriptional regulation of adherens junction disassembly may allow more rapid and effective EMT than a disassembly relying on transcriptional down-regulation of junctional components such as E-Cad. Identifying and characterizing the relevant Sna targets in Drosophila may provide insights into the underlying mechanism for this disassembly, especially with respect to its apparent sensitivity to externally exerted tension. The force-dependent resistance to this Sna function may help in dissecting the underlying molecular functions. Further exploration of Sna’s posttranscriptional effect on junctions and how myosin contraction antagonizes Sna will shed light on understanding of EMT processes (Weng, 2016).
The cytoskeleton is a major determinant of cell-shape changes that drive the formation of complex tissues during development. Important roles for actomyosin during tissue morphogenesis have been identified, but the role of the microtubule cytoskeleton is less clear. This study shows that during tubulogenesis of the salivary glands in the fly embryo, the microtubule cytoskeleton undergoes major rearrangements, including a 90° change in alignment relative to the apicobasal axis, loss of centrosomal attachment, and apical stabilization. Disruption of the microtubule cytoskeleton leads to failure of apical constriction in placodal cells fated to invaginate. This failure is due to loss of an apical medial actomyosin network whose pulsatile behavior in wild-type embryos drives the apical constriction of the cells. The medial actomyosin network interacts with the minus ends of acentrosomal microtubule bundles through the cytolinker protein Shot and disruption of Shot also impairs apical constriction (Booth, 2014).
Echinoid is an immunoglobulin domain-containing transmembrane protein that
modulates cell-cell signaling by Notch and the EGF receptors. In
the Drosophila wing disc epithelium, Echinoid is a component of adherens
junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and
β-catenin (a DE-Cadherin interacting protein) each possess a C-terminal
PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly
position Bazooka to adherens junctions. Echinoid also links to actin filaments
by binding to Canoe/AF-6/afadin. Moreover, interfaces between
Echinoid- and Echinoid+ cells, like those between
DE-Cadherin- and DE-Cadherin+ cells, are deficient
in adherens junctions and form actin cables. These characteristics probably
facilitate the strong sorting behavior of cells that lack either of these
cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin
accumulate a high density of the reciprocal protein, further suggesting that
Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion (Wei, 2005).
Several observations prompted the study of Ed as a canonical CAM in the
monolayered wing imaginal disc. Thus, mitotic recombination clones of cells
mutant for the null allele ed1x5 exhibit rounded and smooth
contours, in contrast to clones of wild-type
cells that show wiggly shapes. This indicated that
ed- /- cells have distinct
adhesive properties and assort with themselves rather than with the surrounding
ed+/- M+/- cells.
(ed1x5 clones were M+,
since without a growth advantage they hardly survive). It was also observed that Ed was absent
from the membrane of the heterozygous cells that contacted the mutant cells,
a finding consistent with the
observation that Ed forms homophilic interactions and that these are required to
incorporate/stabilize Ed at the cell membrane. Finally, Ed was found to
localize basally to the apical marker Crb and apically to the basolateral marker Dlg.
In fact, Ed colocalizes with both DE-Cad and Arm,
and, therefore, it might be part of AJs. AJs are structures important for
cell-cell contact and recognition. So, these results suggested that Ed plays a
role in cell-cell adhesion (Wei, 2005).
Whether Ed affects components of AJs was examined by analyzing the localization of Arm within ed mutant clones. Arm strongly accumulates at the apical membranes of ed- /- cells, and these cells
have a reduced apical surface.
Both effects are clear in small clones, but cells within larger clones (over hundreds of cells) had both
the density of Arm and the apical surface more similar to those of the wild-type
cells. Similar observations were made with DE-Cad and Actin. It is suggested that the increased concentration of these molecules in small clones most probably results from the apical constriction as supported by the accumulation of nonmuscle myosin II, without a net per cell increment of these proteins.
Alternatively, it could result from increased stability of these proteins. The
apical constriction continued through the SJs and ended at the planes just below
the GJs as revealed by an Innexin antibody. Hence, these
ed- /- cells adopt a bottle
shape. In contrast, the apposed ed- /- and
ed+/- cells that form the border of the clone enlarge
and adopte a rectangular shape. At this interface, the
ed- /- cells often contacted the
heterozygous cells by their long sides, as if in an attempt to minimize the number
of cells that formed the interface (Wei, 2005).
Interestingly, Arm and DE-Cad, but not Actin,
are depleted at the interface membrane of both small and large clones. This suggests that ed- /- and ed
heterozygous cells discriminate one another and that AJs do not form properly in
between them (Wei, 2005).
ed clones are surrounded by an Actin 'cable'. High-magnification images
suggest that the cable is contained within the ed heterozygous cells
surrounding the clone and that it is therefore generated by these cells. Several observations
suggest that this Actin cable exerts a force. The cells surrounding an ed
clone elongate toward the clone and accumulate nonmuscle myosin II at the
interface membrane, as if attempting to cover the space exposed by the apically constricted
ed- /- cells. This effect is
reminiscent of the stretching of the leading-edge cells that will cover the
underlying amnioserosa during dorsal closure of the embryos. In the wing disc,
the boundary that separates the dorsal (D) and ventral (V) regions of the wing
pouch has the shape of a smooth arc and contains an actin 'fence'.
After the second instar, this boundary corresponds to a compartment border that
imposes absolute restrictions to cell lineages. Large
ed- /- clones close to or
touching this boundary displace it toward the clones. In contrast, ed clones that
straddle the boundary do not overtly distorted it, although the boundary could be less
smooth within the clone. (Straddling clones
might be originated before the compartment border was established or might be
formed of D and V clones that fuse together). Moreover, the Actin
cable surrounding the clones fuse with the Actin fence at the D/V
boundary, suggesting that the distortion of this boundary is effected through
this Actin linkage. Control
ed+ M+
clones do not induce such distortions. These observations
suggest that the Actin cable may contribute to the roundish shape of the
ed clones and help confine their cells (Wei, 2005).
DE-Cad is a classical homophilic cell adhesion molecule of
AJs. It interacts with β-catenin/Arm, which in turn binds α-catenin.
Through the association between α-catenin and F-Actin, DE-Cad establishes
links between cells that connect to the Actin cytoskeleton. This study shows that Ed
is another CAM that, at the resolution of confocal microscopy, is
also located at the AJs of imaginal disc cells. While cells in clones mutant for
ed still seem to form normal AJs, the cells at the border of the clone
seem impaired in forming them. It is hypothesized that this may help them segregate
from surrounding ed+/- cells. Ed was identified as a
binding partner for PDZ proteins that, similarly to Arm, helps localize Baz to
AJs. Moreover, it was found that through the binding of Cno, Ed, like
DE-Cad/β-catenin, may link to F-Actin. Hence, Ed has functions in
cell-cell adhesion similar to those of DE-Cad (Wei, 2005).
The differential adhesion hypothesis proposes that cell sorting may be driven by differences in
the quantity and/or quality of adhesive molecules displayed on the surface of
cells. In keeping with this hypothesis, it was found that
ed- /- cells sort out from
ed+/- cells, as shown by the remarkably round shapes
and smooth contours of the ed clones. Moreover, their differential
adhesiveness is also manifest by the fusion of different ed clones to
yield composite but still roundish clones. It is suggested that contraction of the
apically enriched Actin network and of the actin cable surrounding the clone,
possibly by interaction with nonmuscle myosin II also present there, may
contribute to the the apical constriction of the
ed- /- cells. It was also observed
that the interface between ed+/- and
ed- /- cells is depleted of
DE-Cad, Arm and Baz, besides completely lacking Ed. This strongly suggests that
this interface is deficient in AJs and probably helps to insulate
ed- /- cells from the
surrounding ed heterozygous cells. It is hypothesized that this deficiency of
AJs, which may reduce adhesion between ed+/- and
ed- /- cells, and
the inward-pulling force generated by apical constriction and the actin cable
may help create the smooth and rounded contour of the clones at the level of
AJs. At the plane of SJs, the clonal boundary is not as smooth. This may be due
to the presence of normal levels of SJs, since seemingly wild-type amounts of
Dlg were detected at the interface membrane. Normal levels of SJs may allow the
clones to remain integrated in the epithelium. It is stressed that when ed
clones grow large, the apical constriction disappears, suggesting that the
forces responsible for this constriction become insufficient or no longer
operate. If the force is exerted, at least in part, by the Actin cable
surrounding the clone, as in a purse-string mechanism, it would make sense that
this force becomes ineffectual as the number of cells within the clone
increases. Remarkably, these differences of apical cell constriction observed in
small and large ed clones have a correlate on the adult wing blade: small
clones display an increased density of trichomes, implying that their cells are
small or more tightly packed, whereas large clones have cells of normal size.
This indicates that the apical constriction is retained through imaginal disc
eversion, when the disc epithelium changes from columnar to planar (Wei, 2005).
In the embryonic
epithelium, Baz, localized to both AJs and the marginal zone, is the initial
apical regulator. How
is Baz recruited to the apical domain? In the follicular epithelium, Baz is
localized to this domain through lateral exclusion mediated by PAR-1/14-3-3 and
apical anchoring by Crb/Sdt/Patj. The data support an additional mechanism to localize
Baz to the apical domain. Both Ed and Arm can bind Baz through their C-terminal
PDZ binding motif and therefore they may redundantly localize Baz to AJs.
Indeed, the localization of Baz to AJs is relatively normal in the absence of
either one. Most Baz is lost only when both Arm and Ed are depleted, as
occurs at the interface membrane of ed clones or in large shg
clones where Ed gradually breaks down. In the latter case, there is good
colocalization between Baz and the sites maintaining residual Ed. It is suggested
that in the epithelium of the wing disc, Baz localizes to AJs by the combined
effects of its binding to Ed/Arm and the lateral exclusion of PAR-1/14-3-3.
Additionally, apical anchoring of Baz may be mediated by direct association
between the Baz and Crb apical complexes. During early embyogenesis, Ed is also
present at pseudocleavage furrows. This observation,
together with the ability of Ed to localize Baz to AJs, may explain the finding
that during cellularization, Baz can accumulate apically in the absence of Arm.
Ed also binds to the PDZ domain of Cno and mediates its localization to AJs, where Cno
interacts with F-Actin either directly or indirectly through the association
with Polychaetoid/ZO-1. Interestingly, the evolutionally conserved EIIV domain
of Ed binds Baz and Cno in a mutually exclusive manner. Thus, the concentrations
of and differential affinities between Ed, Baz, and Cno should determine their
dynamic equilibrium at AJs (Wei, 2005).
Although Baz is critical to form AJs in the
blastoderm and in the follicular epithelium, removal of Baz
(or Par-6) from cells of the wing disc does not affect the
localization of DE-Cad or Ed to AJs. This is consistent with the report that in
imaginal discs, Baz does not affect the localization of DE-Cad and Dlg but is
required for the asymmetric localization of cell fate determinants. Together, these
results suggest that in wing discs, the Baz complex is not critical for the
formation of AJs, and that the effect of the loss of Ed on AJs
formation/maintenance is not due to Baz depletion (Wei, 2005).
Several similarities between the roles of DE-Cad and Ed in the
wing disc epithelium are worth noting. Both Ed and DE-Cad are CAMs that
establish homophilic interactions and localize to AJs. The absence of either Ed
or of DE-Cad in cells of small clones causes their apical constriction and
strong segregation from wild-type cells, giving rise to smooth round borders. In
both cases, the mutant cells are impaired in forming AJs with neighboring
wild-type or heterozygous cells and are surrounded by an Actin cable. Ed
interacts with Cno, and DE-Cad with Arm, and both Cno and Arm directly or
indirectly associate with F-Actin. Thus, Ed and DE-Cad represent two distinct
classes of CAMs, with widely different chemical compositions, that connect to
F-Actin, contribute to cell adhesion in the wing disc, and seem to have
partially overlapping functions (Wei, 2005).
In contrast, DE-Cad and Ed differ in
their ability to regulate the apical/basal cell polarity. Ed affects components
of AJs, but not those of the apical Crb and the basolateral Dlg complexes. In
contrast, DE-Cadherin is necessary for Crb localization, but similarly to Ed, it
is not required for Dlg localization. Furthermore, the maintenance of Ed at AJs
requires DE-Cad. In contrast, localization of DE-Cad to AJs is independent of
Ed. Interestingly, the DE-Cad/Arm complex is not essential for the formation of
the follicular epithelium, but upon removal of this complex, the integrity of the
epithelium is lost slowly over the period of several days. This suggests that
other molecules may be maintaining the epithelial structure. During stages
1 to 10 of oogenesis Ed is mainly expressed in the follicle cells, and these cells,
if mutant for ed, show at low frequency a multilayered structure with
disrupted expression of some polarity markers. Thus,
it will be of interest to elucidate whether, in this epithelium, Ed and
DE-Cad/Arm also play partially redundant roles in cell adhesion and apical/basal
polarity. While both Ed and DE-Cad contribute to cell adhesion and recognition,
it is unclear whether each molecule imparts specific recognition properties to
cells, so that the final cell-cell affinity results from the sum of distinct
affinities mediated by these different CAMs. More specifically, can an increased
level (density) of DE-Cad replace the absence of Ed? The results showing that
ed- /- cells, with either normal
levels (in large clones) or high density (in small clones) of DE-Cad, do not
intermix with wild-type cells suggests that the binding specificity provided by a
given CAM is not overruled by a higher level (density) of a different CAM.
Moreover, the cell sorting properties conferred by Ed cannot account for the
separation of cells at both sides of the A/P compartment boundary of the wing
disc because A and P cells do not intermingle within composite ed, smo
double mutant clones. (Similarly, DE-Cad is not responsible for the sorting out
of A and P cells. Hence, cell-cell adhesion in the wing disc appears to depend on
multiple CAMs (Ed, DE-Cad, etc.), each imparting specific cell recognition
properties. Although Ed and its C-terminal EIIV motif are conserved in
invertebrates, no
clear vertebrate homolog with 7 Ig domains and a PDZ domain binding motif has
been found. Nectin1-4 comprises a family of 3 Ig domain-containing CAM that have
several differentially spliced forms and localize to AJs. Most spliced
forms share a conserved C-terminal E/A-X-Y-V that binds the PDZ domain of
Afadin. Moreover, this motif also interacts with Par-3, the vertebrate homolog
of Baz. In spite of these similarities, overexpression of either nectin 1-α or
3-α does not rescue the remarkable clonal phenotype of ed (Wei, 2005).
Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. This study used the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. Inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/ß-catenin complex, and shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating ß-catenin localization during gland morphogenesis. These results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis (Pirraglia, 2006).
This study shows that the Rac GTPases regulate salivary gland morphogenesis through modulation of cadherin/catenin-based cell–cell adhesion, likely by dynamin-mediated endocytosis. The characterization of the Rac mutant phenotypes suggests a model where Rac normally regulates cadherin-mediated cell–cell adhesion in salivary gland cells to allow enough plasticity for its invagination and migration yet keep the cells of the tube adhered to one another so that the gland can migrate as a cohesive tube. One mechanism by which cell surface cadherin levels are regulated is through selective endocytosis of E-cadherin from the apical–lateral membrane in a dynamin-mediated process. When Rac function is compromised through loss-of-function mutations or expression of dominant-negative mutations, the balance between E-cadherin at the plasma membrane and internalized E-cadherin appears to be abrogated so that more E-cadherin remains at the plasma membrane resulting in increased cell–cell adhesion and causing the gland to sever. These studies reveal the importance of precise regulation of adherens junction remodeling during cell migration in the context of a developing organ (Pirraglia, 2006).
In all stage 14 Rac1L89 mutant embryos examined, the salivary gland broke apart close to its approximate mid-point. Reduction in cadherin levels rescues the mutant Rac severing phenotype, suggesting that severing occurs because loss of Rac leads to an increase in cadherin-mediated cell–cell adhesion. At least two possible explanations for the midpoint severing phenotype are envisioned. In the first scenario, levels of cadherin remodeling may differ throughout the gland such that in Rac1L89 embryos the cells in the distal tip are least affected and cells in the mid-region of the gland are most affected by the increase in cadherin function. In this situation, when the distal cells begin to migrate posteriorly, the increased adhesivity of the mid-region cells prevents their migration and causes the gland to sever in the middle. In the second scenario, movement of the mid-region and the distal region of the gland may occur through different mechanisms. It is possible that while the distal most cells migrate by undergoing cell shape change and extending prominent protrusions in the direction of migration, cells in the middle of the gland may follow the distal cells by rearranging their positions along the gland, such as occurs during the convergence extension movements observed in epithelial morphogenesis. Dynamic remodeling of E-cadherin may be particularly important for proper rearrangement of the mid-region cells and an inability to rearrange when E-cadherin adhesion is increased may cause severing of the gland and subsequent separation of the migrating distal portion from the rest of the gland. Alternatively, it is possible that both of these scenarios are at play during normal salivary gland migration. Currently it is not possible to distinguish between these possibilities. In the developing tracheal tubes, Rac1 is required for cell rearrangements; in tracheal cells expressing a dominant-negative Rac1 mutation, the dorsal branch was shorter than that of wild-type embryos. Therefore, it will be important to determine whether cell rearrangement plays a role during salivary gland migration and to further elucidate the role of the Rac genes in this process (Pirraglia, 2006).
When Rac1 function is over-activate, dynamin-mediated endocytosis of E-cadherin may be increased, resulting in decreased cadherin at the plasma membrane, and decreased cell–cell adhesion. The loss of adhesion leads to the dispersal of salivary gland cells and ultimately cell death. Preventing Rac1V12-induced cell death led to the formation of abnormally shaped glands demonstrating that the Rac1V12 salivary gland phenotype is primarily due to abrogation of gland morphogenesis and not to activation of the apoptotic pathway. Moreover, since wild-type full-length E-cadherin is sufficient to rescue the Rac1V12 salivary gland phenotype, loss of cadherin function appears to be the primary cause for salivary gland defects. Thus, the Rac genes function in salivary gland cells to regulate E-cadherin-mediated cell–cell adhesion during tube morphogenesis (Pirraglia, 2006).
During salivary gland morphogenesis, gland integrity is kept intact while cells perform extensive cell shape changes and movements. Rac-regulated endocytosis of E-cadherin is one mechanism by which cell–cell adhesion is likely to be downregulated temporarily. After E-cadherin is endocytosed, it can be recycled back to the cell surface, sequestered transiently inside the cell or routed to late endosomes and lysosomes for degradation. Once salivary gland migration is complete and the gland has reached its final position, cell–cell adhesion may then need to be strengthened again in the mature gland and Rac activity may be downregulated to promote increase in surface cadherins (Pirraglia, 2006).
In addition to endocytosis, studies in mammalian cultured cells have shown that Rac can regulate levels of cell surface E-cadherin by other mechanisms, such as cleavage by presenilins and metalloproteinases, or tyrosine phosphorylation of the cadherin adhesion complex in a process involving reactive oxygen species. Thus, it will be interesting to determine whether additional mechanisms of E-cadherin regulation exist in salivary gland cells during gland morphogenesis (Pirraglia, 2006).
Numerous studies in cell culture have demonstrated that recycling of E-cadherin occurs in both a clathrin-dependent and caveolin-dependent manner. Since dynamin mediates both clathrin- and caveolin-dependent endocytosis, these studies do not allow distinguishing which type is involved in cadherin endocytosis during salivary gland migration. Alternatively, both types of endocytosis may mediate Rac1 regulation of E-cadherin in salivary gland cells (Pirraglia, 2006).
Expression of the Rac1V12 mutation in salivary gland cells leads to loss of expression of salivary gland specific proteins, apical–basal polarity proteins and E-cadherin/β-catenin. Concomitant with changes in gene expression, Rac1V12 mutant salivary gland cells lose adhesion to each other and subsequently migrate away or die by apoptosis. The data suggest that overactivation of Rac1 primarily affects E-cadherin/β-catenin-mediated adhesion and salivary gland cell fate and that the observed cell death is a secondary consequence of these earlier changes. When cell death was prevented in Rac1V12 embryos by expressing p35, more cells expressed the salivary gland specific protein PH4αSG1 than in Rac1V12 embryos; however, the expression level was drastically reduced compared to wild-type, suggesting that even in the Rac1V12p35 cells, cell differentiation was still mostly altered. Moreover, Rac1V12p35 salivary gland cells did not form a normal gland, demonstrating a role for Rac1 in gland morphogenesis. It is possible that apoptosis of Rac1V12 cells is brought about by the loss or reduction of Forkhead (Fkh) function. Fkh is expressed early in the salivary gland placode and its expression is maintained throughout embryogenesis. In the absence of fkh function, salivary gland cells die by apoptosis during the invagination stage. Since expression of dCREB-A and PH4αSG1 is reduced in Rac1V12 mutant salivary gland cells, it is possible that Fkh expression is also similarly reduced, thereby, causing the cells to undergo apoptotic cell death (Pirraglia, 2006).
Many human cancers are due to epithelial-derived tumors. When epithelial cells metastasize, they first undergo an epithelial to mesenchymal transition (EMT) before migrating away from the primary tumor to invade surrounding tissues. EMT is characterized by the loss of epithelial polarity and cell–cell adhesion. When Rac1V12 was expressed in salivary gland cells, expression of apical membrane proteins, Crumbs and aPKC and adherens junction proteins E-cadherin and β-catenin, was either lost or mislocalized. Based on these criteria, activation of Rac1 function induces features characteristic of early changes in EMT and metastasis. Interestingly, the expression levels of Rho GTPases are found to be elevated in a number of human cancers. For example, increased Rac protein levels and fast-cycling Rac mutations have been correlated with colorectal and breast tumors. Expression of constitutively active Rac1 causes some salivary gland cells to lose polarity and adhesion to neighboring cells and migrate away in a manner similar to EMT. These findings suggest that Rac1-regulated endocytosis of E-cadherin in the Drosophila salivary glands may be critical in maintaining epithelial character and preventing the loss of cell–cell adhesion and cell polarity. The Drosophila salivary gland might thus be powerful as a simple system to identify and characterize mechanisms that regulate cadherin-based cell–cell adhesion and certain aspects of EMT (Pirraglia, 2006).
Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. This study identifies an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. Two polarized cues have been identified that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. Btsz binds to the Ezrin-Radixin-Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, these data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia (Pilot, 2006).
Homotypic binding of the cell-adhesion molecule E-cadherin (E-cad) at the adherens junctions of epithelial cells organizes the formation of multiprotein complexes, composed in part of the ß-catenin and alpha-catenin proteins, and their dynamic interaction with actin filaments (F-actin). F-actin is required to stabilize E-cad-ß-catenin-alpha-catenin complexes. Moreover, E-cad regulates its own stability through the organization of actin filaments through alpha-catenin: alpha-catenin binds Formin (also known as Diaphanous) and suppresses branching by competing with Arp2/3 (Drees, 2005). When epithelia form through the mesenchymal-epithelial transition, the sites of initial cell contact serve as spatial landmarks for the recruitment of E-cad-ß-catenin-alpha-catenin complexes during the formation of adherens junctions. In primary embryonic epithelia, however, adherens junctions do not form through specific cell contacts, and the spatial cues positioning the adherens junctions in the AJR are less characterized and may be different. The identification of such spatial cues and the mechanisms whereby these cues organize structural, cytoskeletal components associated with the formation and/or stabilization of adherens junctions is an important challenge (Pilot, 2006).
This problem was addressed in the early Drosophila embryo. Formation, stabilization and remodelling of adherens junctions occur in a tightly and genetically controlled sequence involving e-cad (or shotgun), armadillo (or ß-catenin), par-6 , par-3 (or bazooka, baz), aPKC, crumbs and others. A microarray-based RNA interference (RNAi) screen of epithelial morphogenesis identified btsz, a gene previously known to control growth in adult flies (Serano, 2003), as a regulator of embryonic epithelial integrity. In embryos injected with double-stranded RNA (dsRNA) probes specific for btsz (hereafter called btszRNAi embryos), gastrulation is severely affected and the epithelium fails to extend properly. Defects are either strong or medium; that is, they are visible at the beginning of gastrulation or about 15 min later, respectively. The defects are penetrant (80%) and dose dependent. Four different, nonoverlapping probes produce these defects and embryos were not affected with control probes (Pilot, 2006).
Btsz is the only Drosophila member of the synaptotagmin-like protein (SLP) family characterized by the presence of tandem carboxy-terminal C2 boxes. btsz encodes several isoforms (Serano, 2003). In early embryos, btsz1 was not detected but btsz2 and btsz3 are expressed together with btsz0, another isoform not previously reported. At least one of these isoforms is maternally and zygotically provided. The most efficient dsRNA probes used recognizes all three maternally and zygotically expressed isoforms. These isoforms were strongly reduced in btszRNAi embryos, suggesting that RNAi produces a severe btsz loss-of-function phenotype (Pilot, 2006).
Two btsz alleles have been described (Serano, 2003): btszK13-4 introduces a deletion in the amino terminus of btsz2 (residues 501-1,494), btszJ5-2 corresponds to a frameshift mutation that introduces a stop codon at amino acid 390, which leads to a truncation in Btsz0 and Btsz2, and probably the absence of Btsz3. btszK13-4 homozygous female escapers can be recovered and were crossed to heterozygous btszK13-4 or btszJ5-2 males. Although many embryos were not fertilized, those that were reached cellularization and showed epithelial defects during gastrulation: 26% of btszK13-4/btszK13-4 and 46% of btszK13-4/btszJ5-2 embryos. btszJ5-2 germline clones do not produce eggs and btszJ5-2 is lethal. Trans-heterozygous embryos were examined with a deficiency removing the btsz locus (Df(3R)Exel6275, called Dfbtsz): 12% of embryos from crosses of Dfbtsz/btszK13-4 females and wild-type males showed epithelial defects. This proportion went up to 39% when males were heterozygous
btszK13-4/+. It is concluded that btsz is zygotically and maternally required. Whereas RNAi targeted all three btsz isoforms, btszK13-4 left intact a large fraction of Btsz2 and Btsz0, probably explaining the weaker penetrance of phenotypes in btszK13-4 (26%) or btszK13-4/btszJ5-2 (46%) mutants, as compared with btszRNAi embryos (80%). Notably, despite its essential role in the formation of epithelia in early embryos, the recovery of adult escapers suggests that btsz may be dispensable in adult epithelia (Pilot, 2006).
Overexpression of a btsz2 isoform lacking the 3' untranslated region (UTR) rescues the phenotypes produced by an RNAi probe targeting the 3' UTR of all btsz isoforms. Overexpression of btsz2 more robustly rescues the btszRNAi phenotype than does btsz3 overexpression, suggesting that btsz2 has a prominent role. The injection of morpholino antisense oligonucleotides (morpholinos) specific to each btsz isoform confirmed this: a control morpholino showed no defect, a mix of btsz0, btsz2 and btsz3 morpholinos caused penetrant defects (92%), and a btsz2-specific morpholino alone caused defects in 73% of embryos. Experimental focus was therefore placed on Btsz2, a 286-kDa protein (2,645 residues) (Pilot, 2006).
The expression of a Glu-epitope-tagged variant of Btsz2 (Btsz2-Glu) was strongly reduced in btszRNAi embryos. The epithelium failed to maintain its regular morphology in btszRNAi embryos, btsz mutants and btsz morphants. Although cellularization proceeds similarly in btszRNAi and control embryos, at the onset of gastrulation the epithelium collapses and becomes multilayered in btszRNAi and btszK13-4/btszJ5-2 mutant embryos, as compared with controls. A similar phenotype was observed in e-cadRNAi embryos. Thus, btsz controls the stable architecture of primary embryonic epithelia (Pilot, 2006).
These data suggested that btsz might regulate the formation of adherens junctions. In contrast to the wild type, in which E-cad is uniformly present at the adherens junctions, E-cad expression is heterogeneous and the adherens junctions appears severely fragmented in btsz mutants and btszRNAi embryos. Time-lapse recordings of E-cad fused to green fluorescent protein (GFP) showed that adherens junctions forms properly in the AJR of btszRNAi embryos but that, subsequently, E-cad-GFP expression disappears, suggesting a defect in the stabilization but not targeting of E-cad. E-cad-GFP, or endogenous E-cad, disappears in small patches at cell contacts, pointing to defects in actin organization. Indeed, the actin belt in the AJR is fragmented in btszRNAi embryos. Tested were performed to see whether actin organization or the E-cad-ß-catenin-alpha-catenin complexes was the primary cause of the disassembly of adherens junctions in btszRNAi embryos. In e-cadRNAi embryos, in which E-cad was undetectable in the nascent AJR, the actin belt is not considerably affected during early gastrulation and clearly less affected than in btszRNAi embryos at the same stage. Subsequently, however, F-actin was disorganized in e-cadRNAi embryos. This suggests that Btsz is part of an E-cad-independent pathway controlling actin organization in the AJR and consequently junction stability (Pilot, 2006).
Next, Btsz2 localization was examined. Btsz2-Glu is a functional protein that rescues the btszRNAi phenotype. Btsz2-Glu was previously reported to localize apically in follicular epithelial cells (Serano, 2003). In early embryos, Btsz2-Glu is detected at the AJR together with E-cad from the end of cellularization until about 30 min into gastrulation. Subsequently, Btsz2-Glu was found in a subapical compartment. At these early stages, E-cad colocalizes with Par-3 (also known as Baz). Therefore the possible role of E-cad and Par-3/Baz in Btsz2 localization in the AJR was addressed. In e-cadRNAi embryos, the recruitment of E-cad in the AJR is blocked and Btsz2 is normal; by contrast, in par-3/bazRNAi embryos Btsz2 is largely cytoplasmic, like PatJ, another marker of AJR at this stage. Btsz2 is thus a target of the early polarity marker Par-3/Baz, which is required for E-cad localization in the AJR (Pilot, 2006).
The role of the two C2 boxes (C2AB) in the localization of Btsz2 was tested. Purified glutathione S-transferase (GST)-tagged C2AB binds to phosphatidylinositol mono- and bisphosphate species in a Ca2+-dependent fashion in vitro. The in vivo relevance of this binding was assessed. A tagged form of Btsz2 lacking the C2 boxes (Btsz2-DeltaC2-HA) expressed in gastrulating embryos was cytoplasmic and failed to localize at the AJR. Conversely, an epitope-tagged form of C2AB (C2AB-HA) localizes at the plasma membrane in gastrulating embryos. Notably, C2AB is polarized and concentrates in the apical surface and in the AJR. Of all the phosphoinositides that C2AB binds in vitro, phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) is the most abundant at the plasma membrane, suggesting that PtdIns(4,5)P2 could be required for Btsz2 localization in the AJR. Injection of cellularizing embryos with neomycin, a compound that binds and inhibits PtdIns(4,5)P2, resulted in epithelial defects similar to btsz, par-3 or e-cadRNAi, and inhibits the recruitment of Btsz2 at the plasma membrane and the AJR. A fusion between GFP and the pleckstrin homology (PH) domain of phospholipase Cdelta (PLCdelta), which specifically binds PtdIns(4,5)P2, localizes apically and in the AJR, similar to the Btsz C2 boxes. It is concluded that PtdIns(4,5)P2 is a polarized spatial cue required for localization of Btsz in the AJR, and hence for adherens junction stability, together with Par-3/Baz (Pilot, 2006).
How does localized Btsz organize F-actin in the AJR? A large-scale two-hybrid screen identified an interaction between Btsz and Moesin, the only Drosophila member of the Ezrin-Radixin-Moesin (ERM) family of F-actin binding proteins that has been implicated in various aspects of epithelial polarity. This interaction was confirmed and Btsz was shown to bind to the third F3 subdomain of Moesin. A minimal region in Btsz that binds Moesin was narrowed down. This interaction occurred in GST pull-down assays of Drosophila S2 cell lysates and embryonic extracts. The functional relevance of this interaction was assessed. Moesin and the phosphorylated active form of Moesin, which binds F-actin, localizes in early embryos in the apical surface and in the AJR, together with Btsz2 and E-cad, suggesting that the interaction between Btsz and Moesin may spatially define a domain of actin organization in the AJR required to stabilize E-cad. In agreement with this, in btszRNAi and btsz mutant embryos, Moesin localization was diminished in the AJR as compared with controls, and E-cad and Moesin segregated in distinct domains as E-cad progressively disappeared (Pilot, 2006).
Whether Moesin is required for epithelial stability was tested in early embryos. Moesin has a major maternal contribution and is a very stable protein. Moreover, females whose germline is mutant for moesin do not lay eggs. Thus, no phenotype was identified using either various moesin mutant alleles or RNAi. Therefore a dominant-negative construct of Ezrin, a mammalian Moesin orthologue that lacks the C-terminal actin-binding domain and acts as a dominant-negative in Drosophila (EzrinDN, containing residues 1-280) was expressed in early embryos . Embryos expressing EzrinDN during gastrulation showed epithelial defects (41% of embryos) similar to btsz mutants. In particular, cellularization was normal, adherens junctions formed properly, but E-cad was no longer present homogeneously around the AJR (Pilot, 2006).
These results shed light on the mechanisms underlying the spatial control of actin filament and the stability of the adherens junctions in the Drosophila primary embryonic epithelium. In Btsz, an E-cad independent pathway has been identified that participates in F-actin organization in the AJR, together with Moesin. Btsz and Moesin bind and colocalize in the AJR in a btsz-dependent fashion, and expression of a mutant form of Ezrin that does not bind F-actin disrupts adherens junctions stability similar to loss of btsz. Notably, this work identifies upstream polarity cues (Par-3/Baz and PtdIns(4,5)P2) that control the spatial order of actin organization at the AJR through the localization of Btsz. The fact that PtdIns(4,5)P2 acts as a key regulator of epithelial polarity in the early embryo raises the issue of how PtdIns(4,5)P2 metabolism is spatially regulated in epithelial cells. The observation that Par-3 binds PTEN, which converts PtdIns(3,4,5)P3 into PtdIns(4,5)P2, suggests that Par-3/Baz may be part of this process. Thus a key intermediate between polarity cues and structural elements of adherens junctions important for embryonic epithelial stability has been identified. Five SLPs and two SLP-related (Slac2) proteins are close orthologues of Btsz in mammals. It would be worth investigating their potentially conserved role in the dynamic organization of actin at adherens junctions in embryonic epithelia (Pilot, 2006).
A fundamental requirement during organogenesis is to preserve tissue integrity to render a mature and functional structure. Many epithelial organs, such as the branched tubular structures, undergo a tremendous process of tissue remodelling to attain their final pattern. The cohesive properties of these tissues need to be finely regulated to promote adhesion yet allow flexibility during extensive tissue remodelling. This study reports a new role for the Egfr pathway in maintaining epithelial integrity during tracheal development in Drosophila. The integrity-promoting Egfr function is transduced by the ERK-type MAPK pathway, but does not require the downstream transcription factor Pointed. Compromising Egfr signalling, by downregulating different elements of the pathway or by overexpressing the Mkp3 negative regulator, leads to loss of tube integrity, whereas upregulation of the pathway results in increased tissue stiffness. Regulation of MAPK pathway activity by Breathless signalling does not impinge on tissue integrity. Egfr effects on tissue integrity correlate with differences in the accumulation of markers for cadherin-based cell-cell adhesion. Accordingly, downregulation of cadherin-based cell-cell adhesion gives rise to tracheal integrity defects. These results suggest that the Egfr pathway regulates maintenance of tissue integrity, at least in part, through the modulation of cell adhesion. This finding establishes a link between a developmental pathway governing tracheal formation and cell adhesiveness (Cela, 2006).
This study documents a new role for the Egfr pathway in the regulation of
tissue integrity. This new requirement could depend on the described early
peak of Egfr activity, which would be sufficient to prevent defects at later
stages. However, it is proposed that Egfr-promoted epithelial integrity
depends on a later, or continuous but lower, or basal activity of the pathway
that does not correlate with detectable ERK phosphorylation. Consistent with
this hypothesis, it was found that downregulation of the pathway by overexpressing
801 or UAS-EgfrDN with btlGal4, which is
expressed after the early peak of ERK phosphorylation, produces a conspicuous
branch integrity phenotype. In any case, tissue integrity defects are mainly
observed in the most dorsal and ventral tracheal branches, which are subjected
to stronger pulling forces as development proceeds, and, therefore, it is
precisely at late stages when defects in tissue integrity are expected (Cela, 2006).
AJs connecting epithelial cells dynamically disassemble and reassemble,
thereby allowing tissue remodelling. Tracheal tissue remodelling might require
the fine-tuning of cell adhesion properties, since tracheal cells need to be able
to change their relative position (probably by loosening cell adhesion) while
maintaining epithelial continuity. The data indicates that the Egfr pathway is
a modulator of this balance, not only in the tracheal system, but also in
other tissues undergoing extensive remodelling, such as the salivary glands,
where a similar regulation of DE-cad and actin levels is found upon modulation
of Egfr signalling. Conversely, no such a regulation was found in more static tissues, like the ectoderm, whose maintenance was proposed to depend on the maternally provided DE-cad protein. It is suggested that the Egfr pathway plays a role in the modulation of cell adhesion in tissues that undergo dramatic morphogenetic events, which might need the zygotic DE-cad contribution and a more dynamic regulation of cell adhesion. The results indicating a modulation of junctional complexes and/or the actin cytoskeleton by the Egfr pathway establish a link between a developmental pathway required for many biological events and cell
biology in terms of cell adhesiveness and cell shape (Cela, 2006).
The results show that downregulation of several intracellular elements of
the MAPK pathway produce defects in branch integrity, whereas a constitutively
activated form of rl (rlsem) rescues the
phenotype of btlGal4 801 embryos. This suggests that the conserved
MAPK cassette is required to maintain branch integrity (Cela, 2006).
Two tyrosine kinase receptors, Egfr and Btl, activate the MAPK pathway
during embryonic tracheal development. However, the two
receptors, acting through the same intracellular cascade, elicit different
responses. The MAPK pathway requirement in primary branching is likely to
depend on input by btl, whereas the tissue integrity requirement is
likely to depend on input by Egfr. How does the same MAPK pathway
trigger distinct outcomes depending on the receptor that activates it? A
temporal and/or spatial differential activation of the MAPK pathway could
account for the different outcome. In addition, differences in the composition
of the intracellular cascade due to specific transducers for one type of
receptor, such as downstream of FGFR (dof;
stumps-FlyBase), could contribute. Finally, quantitative and/or qualitative
differences in the activation of the intracellular transducers by the
different receptors could also underlie the outcome diversity (Cela, 2006).
Similar to these observations, air sac development in Drosophila has
been recently reported to require both Btl and Egfr, and each receptor seems
to elicit different responses. Furthermore, since during embryonic
tracheal development, an uncoupling of the MAPK cassette and pnt has
been observed during air sac development.
These parallels suggest a common mechanism for generating different responses
from the same intracellular transduction pathway (Cela, 2006).
The loss of tissue continuity and cell detachment observed in Egfr
downregulation conditions may be due, at least in part, to a decrease in cell
adhesion. Accordingly, a mild, but reproducible, decrease is observed in the
accumulation of DE-cad and cortical actin. As inferred from the phenotypes,
such a mild decrease could cause a loss of cell adhesion during tracheal
remodelling, while not grossly affecting other processes requiring
DE-cad-based cell adhesion, such as branch fusion. As expected, it was found that compromising AJ assembly or the
actin cytoskeleton also gives rise to defects in tracheal tissue
integrity (Cela, 2006).
Cadherins have been shown to support cell cohesion and participate in
morphogenetic events. The actin cytoskeleton also plays an important role in
shaping the cell architecture and in many morphogenetic processes. AJs and the
actin cytoskeleton are intimately coupled, and their formation and maintenance
is interdependent. Such interdependence is also observed in the tracheal
system (Cela, 2006).
Cadherin-based cell-cell adhesion can be regulated at transcriptional and
posttranscriptional levels. The modulation of a DE-cadGFP chimaera
driven by heterologous promoters shows that, in the current case, DE-cad regulation is posttranscriptional. Several posttranscriptional mechanisms of DE-cad
regulation have been proposed, and a role for the Egfr pathway can be envisaged in each of them. A first mechanism
is at the level of DE-cad endocytic trafficking. In this context, the Egfr
pathway could modulate the balance between recycling to the plasma membrane of
internalised DE-cad or lysosomal targetting and degradation. A second
mechanism of cell-cell adhesion regulation is posttranslational modifications
of AJ components, such as phosphorylation or ubiquitination. Finally, another
possible mechanism of regulation is through the cytoskeleton. The Rho family
of small GTPases plays a key role in actin cytoskeleton regulation, and growth
factor receptors such as Egfr have been reported to regulate their activity. Remarkably, the Egfr pathway has been recently shown to
regulate the expression of the rhoGAP cv-c in the tracheal placodes, and
it was found that cv-c mutants display tracheal integrity defects,
although they are milder than those seen upon downregulation of the Egfr
signal. It is therefore proposed that cv-c is at least one of the
effectors of Egfr-mediated modulation of DE-cad levels and tracheal tissue
integrity. Further analysis will be needed to disentangle the exact molecular
mechanisms and to find other possible mediators of the Egfr signal (Cela, 2006).
The decrease of cadherin activity upon activation of the Egfr pathway has
been extensively reported in the literature. This study reports the opposite: that Egfr pathway downregulation correlates with a decrease of cadherin-based cell adhesion. Although this is not the first example of such a relationship, it illustrates the versatility and complexity of the interactions occurring between signalling pathways and adhesion molecules, and establishes another model with which to analyse how cell adhesion is modulated (Cela, 2006).
Force generation by Myosin-II motors on actin filaments drives cell and tissue morphogenesis. In epithelia, contractile forces are resisted at apical junctions by adhesive forces dependent on E-cadherin, which also transmits tension. During Drosophila embryonic germband extension, tissue elongation is driven by cell intercalation, which requires an irreversible and planar polarized remodelling of epithelial cell junctions. This study investigate how cell deformations emerge from the interplay between force generation and cortical force transmission during this remodelling in Drosophila melanogaster. The shrinkage of dorsal-ventral-oriented ('vertical') junctions during this process is known to require planar polarized junctional contractility by Myosin II. This study shows that this shrinkage is not produced by junctional Myosin II itself, but by the polarized flow of medial actomyosin pulses towards 'vertical' junctions. This anisotropic flow is oriented by the planar polarized distribution of E-cadherin complexes, in that medial Myosin II flows towards 'vertical' junctions, which have relatively less E-cadherin than transverse junctions. The evidence suggests that the medial flow pattern reflects equilibrium properties of force transmission and coupling to E-cadherin by α-Catenin. Thus, epithelial morphogenesis is not properly reflected by Myosin II steady state distribution but by polarized contractile actomyosin flows that emerge from interactions between E-cadherin and actomyosin networks (Rauzi, 2010).
The data suggest that the anisotropic actomyosin flow may largely depend on the distribution of junctional anchoring points. This requires E-cadherin/β-Catenin complexes at AJs and depends on α-Catenin. E-cadherin/β-Catenin/α-Catenin complexes are planar polarized, such that medial pulses flow towards regions with lower amounts of E-cadherin complexes. The level of E-cadherin along 'vertical' relative to adjacent junctions (E-cadherin anisotropy) is also fluctuating. Moreover, the onset of medial pulses coincided with the time when E-cadherin anisotropy reached a local maximum raising the possibility that E-cadherin anisotropy may orient the actomyosin flow. Reduction of E-cadherin by RNAi causes the disappearance of medial Myo-II. The junctional Myo-II level is consequently strongly reduced and no longer planar polarized. It was reasoned that reducing the levels of α-Catenin by RNAi should attenuate coupling more subtly. α-Catenin RNAi reduces the number of E-cadherin clusters at AJs and disrupts interactions with junctional F-actin. Moreover, the distribution of E-cadherin is no longer planar polarized in α-CateninRNAi embryos. This is associated with a loss of medial and junctional Myo-II planar polarity. Thus, the planar polarized distribution of E-cadherin/β-Catenin/α-Catenin complexes biases the flow of medial Myo-II and junctional polarization (Rauzi, 2010).
In addition to Myo-II contractility, flow requires (1) crosslinkers between filaments to transmit tension within the medial meshwork, and (2) coupling at the cortex to E-cadherin/β-Catenin/α-Catenin complexes. Increased levels of E-cadherin in 'transverse' junctions may change properties of the actin network (for example, crosslinking/viscosity) and inhibit internal transmission of contractile forces and hence prevent D-V oriented flow. To test this, the force balance within the medial actomyosin network was disrupted by focal ablation, and the redistribution of medial clusters was imaged. If increased E-cadherin levels at transverse junctions inhibit tension transmission along the D-V axis, then medial pulses should not flow in this direction following ablation. However, it was observed that Myo-II medial clusters flowed radially and away from the point of ablation towards the junctions in 100% of cases, even towards transverse junctions. Focal ablation of the actin meshwork produces a local hole, which expands radially. This argues that transverse junctions do not inhibit flow per se and that flow directionality emerges from the properties of the actomyosin meshwork integrated over the entire apical surface (Rauzi, 2010).
The mechanical properties of the medial actomyosin network are locally defined by Myo-II contractility (concentration, affinity, duty cycle), tension transmission within the network (crosslinking), and viscous resistance to deformations (interactions between filaments). Moreover, these properties fluctuate owing to protein turnover and interactions. E-cadherin is known to anchor and modify actin dynamics. The results suggest that the polarized distribution of E-cadherin may control the actomyosin flow pattern by spatially modulating mechanical properties of the actin network (Rauzi, 2010).
Current models of epithelial morphogenesis centre on Myo-II steady state distribution and associated contractile forces. The current data show however that cell deformations cannot be simply derived from the Myo-II distribution itself, but from two central features of actomyosin dynamics, namely concentration (pulses) and movement (flow). Pulsed dynamics defines the rhythm and possibly the speed of deformation. Flow pattern, which in the case of intercalation is anisotropic, dictates the orientation of cell deformation. Flows of Myo-II foci have been reported in the one-cell stage C. elegans embryo, pointing to a more general property of actomyosin networks. An important future avenue of research will be to investigate what properties of actin networks control Myo-II flow dynamics in different systems (Rauzi, 2010).
Some organs in animals display left-right (LR) asymmetry. To better understand LR asymmetric morphogenesis in Drosophila, LR directional rotation of the hindgut epithelial tube was studied. Hindgut epithelial cells adopt a LR asymmetric (chiral) cell shape within their plane, referred to as planar cell-shape chirality (PCC). Drosophila E-cadherin (DE-Cad) is distributed to cell boundaries with LR asymmetry, which is responsible for the PCC formation. Myosin ID (Myosin 31DF) switches the LR polarity found in PCC and in DE-Cad distribution, which coincides with the direction of rotation. An in silico simulation showed that PCC is sufficient to induce the directional rotation of this tissue. Thus, the intrinsic chirality of epithelial cells in vivo is an underlying mechanism for LR asymmetric tissue morphogenesis (Taniguchi, 2011).
Directional left-right (LR) asymmetry is widely found in animals, such as in the position and structure of the heart, spleen, gut, and lung in vertebrates. The mechanisms of LR axis formation are well understood in some vertebrates, and the cellular basis for LR symmetry breaking, including cell polarities, is beginning to be elucidated. Drosophila shows a directional LR asymmetry of certain organs, including the embryonic hindgut. Although some unique features of Drosophila laterality development have been revealed, such as the involvement of myosin ID (MyoID), the detailed mechanisms of its LR asymmetric development remain largely unknown (Taniguchi, 2011).
The Drosophila embryonic hindgut begins as a symmetric midline structure that curves ventrally at stage 12. It subsequently makes a 90° left-handed rotation, forming a rightward curving structure by stage 13. The hindgut epithelium, but not the overlying visceral muscles, is responsible for this rotation, which is not accompanied by cell proliferation or cell death. Therefore, it was speculated that the hindgut epithelial cells themselves might have LR polarity, which could contribute to the rotation (Taniguchi, 2011).
To analyze LR polarity in the hindgut epithelial cells, the
locations of the centrosomes, which reflect cell polarity in other
systems, was examined. Each cell's centroid was calculated with respect to its boundaries and the relative position of the centrosome was plotted, labeled with green fluorescent protein (GFP)-centrosomin. In wild-type animals, the relative position of the centrosome was significantly biased to the right-posterior region. These results suggest that hindgut epithelial cells adopt a LR polarity within their plane before the hindgut rotates (Taniguchi, 2011).
It was speculated that this LR polarity would be reflected in the cell shape
and participate directly in the left-handed rotation. To address this,
the angle was measured between apical cell boundaries and the
antero-posterior (AP) axis of the hindgut epithelial tube before
rotation (late-stage 12). These apical cell boundaries corresponds to the zonula adherens (ZA). Cell-boundary angles of -90° to 0° to the AP axis were more frequent than those of 0° to 90°, indicating that hindgut epithelial cells have a LR-biased planar cell shape. This LR bias was designated as planar cell-shape chirality (PCC), because the mirror image of the cell's planar shape does not overlap with its original cell shape (Taniguchi, 2011).
Previous studies demonstrated that the hindgut rotates right-handedly in embryos homozygous for Myo31DF, which encodes MyoID. In Myo31DFL152 homozygotes, the distribution of angle x° was reversed from that of wild type, although the LR bias became less prominent. The reversed PCC in Myo31DFL152 was rescued by the overexpression of Myo31DFGFP. Rho family guanosine triphosphatases, including Rho1 and Rac1, regulate the organization of the actin cytoskeleton. It was previously shown that overexpression of a dominant-negative Rho1 (Rho1.N19) or Rac1 (Rac1.N17) in the hindgut epithelium disrupts the hindgut's LR asymmetry. No PCC was observed in these epithelial cells, suggesting that PCC formation depends on the actin cytoskeleton. These results support the suggestion that PCC could determine the subsequent laterality of the hindgut (Taniguchi, 2011).
To identify genes involved in PCC formation, a screen was carried out for mutations affecting LR asymmetry of the hindgut. It was found that shotgun (shg) mutations (shgR758, shgR1232, and shgR69, a null allele) disrupted the laterality of the hindgut. shg encodes DE-Cad, a conserved transmembrane protein that mediates cell-cell adhesion in the epithelium. Genetic analyses suggested that DE-Cad functions downstream of MyoID, and both are required in the hindgut epithelium just before its rotation for normal LR asymmetric development. In shgR69 homozygotes, the angle x° did not demonstrate LR asymmetry, indicating that PCC was not formed in this mutant. This PCC defect in shgR69 homozygotes was rescued by the overexpression of shgDECH (Taniguchi, 2011).
To understand how DE-Cad contributes to PCC formation, whether the distribution of DE-Cad showed LR polarity in hindgut epithelial cells was examined. For this, the mean of DE-Cad's relative intensity at the ZA of each cell boundary in the hindgut epithelium was at late-stage 12 was calculated. In wild type, the mean intensity was significantly greater at the cell boundaries with an angle x° of -90° to 0° than in those with 0 to 90° angles, whereas this situation was reversed in Myo31DFL152 homozygotes. Rose diagrams depicting the intensity of DE-Cad in the cell boundaries bundled for 30° intervals showed that DE-Cad was enriched in cell boundaries with an angle x° of -90° to -30°. Conversely, in Myo31DFL152 homozygotes, this situation was reversed. This reversed bias was restored to the wild-type situation by overexpressing Myo31DFGFP in the hindgut epithelium (Taniguchi, 2011).
It was then asked whether the LR bias of DE-Cad distribution was attributable to a cell-autonomous function of MyoID. To address this, a new system was developed for generating a mosaic hindgut epithelium composed of Myo31DFL152 homozygous cells with (+) or without (-) Myo31DFmEGFP overexpression. In the hindgut epithelium, the cell boundaries were classified into three types according to the cell type on either side: +/+, green; +/-, yellow; -/-, magenta. Cell boundaries of +/+ showed the wild-type LR bias of DE-Cad localization, which was reversed in the -/- boundaries. The +/- cell boundaries did not show a statistically significant LR bias. Thus, the LR asymmetry of DE-Cad distribution at each cell boundary is attributable to the concordance of LR polarity in two adjacent cells (Taniguchi, 2011).
To gain insight into how MyoID reverses the LR asymmetric distribution of DE-Cad, defects were sought in endocytic trafficking, because DE-Cad's localization to the ZA is controlled by its recycling. Rab11-positive recycling endosomes became fewer in the apical-middle part of cells in Myo31DFL152 homozygotes compared with wild type, and this defect was restored by Myo31DFGFP expression. These results may suggest that MyoID is involved in the recycling of DE-Cad (Taniguchi, 2011).
Besides the angle x°, the length was also measured of cell boundaries at the ZA of the hindgut epithelial cells. In wild-type animals, the cell boundaries gradually expanded from late-stage 12 to late-stage 13. In homozygous Myo31DFL152 or shgR69 embryos, the length was greater than in wild type at all stages examined. This increase was rescued by the overexpression of Myo31DFGFP or shgDECH in the respective mutant background. Thus, DE-Cad and MyoID appear to restrict the expansion of these cell boundaries, suggesting that these proteins introduce cortical tension, possibly with LR asymmetry (Taniguchi, 2011).
To evaluate the idea that PCC is involved in the hindgut LR asymmetric development, an in silico simulation model was built of the PCC of the hindgut epithelial cells and the directional rotation of the tube composed of these cells. This model consisted of two epithelial sheets composed of model cells, forming the dorsal and ventral arcs of a tube with boundary cells separating the sheets, as found in vivo. In this simulation, the number of cells along the AP and LR sides was set to mimic the in vivo situation, and a statistical LR shape bias was not introduced initially. In vivo, DE-Cad was enriched at cell boundaries with an angle x of -90° to 0° and might restrict the cell-boundary expansion. Therefore, in this simulation, the constriction of cell boundaries was maximized at -45° to the AP axis of the hindgut epithelial tube, and the maximized value was twofold greater than at -135° or +45°. This parameter introduced PCC in the modeled epithelial cells (corresponding to late-stage 12) (Taniguchi, 2011).
Because DE-Cad and MyoID were required before but not during the left-handed rotation of the hindgut epithelium, no LR bias was added to the cell-boundary constriction during the epithelial remodeling. The removal of LR bias subsequently led the modeled epithelial cells to assume stable cell shapes that were mostly regular hexagons (corresponding to late-stage 13). This progressive transition in cell shape was also observed in vivo. This simulation reproduced the 90° left-handed rotation of the epithelial tube in silico, suggesting that PCC is sufficient to explain this rotation in vivo. In addition, LR asymmetric changes in the cell-boundary length observed in the hindgut epithelium in vivo were recapitulated in the simulation, supporting the validity of the model (Taniguchi, 2011).
This study has reported PCC as a previously unknown mechanism of LR asymmetric
morphogenesis. Various mutants of genes encoding the core components of planar cell polarity (PCP), a well-understood type of epithelial planar polarity, did not affect the laterality of the Drosophila embryonic gut, suggesting that PCC is not simply a variant of PCP. Although the importance of single-cell chirality has not been studied in multicellular organisms in vivo, intrinsic cell chirality has been found in the LR-polarized protrusion of neutrophil-like cells in vitro. Therefore, cell chirality may be a general property of animal cells. These findings demonstrate a contribution of such chirality to LR asymmetric morphogenesis (Taniguchi, 2011).
E-cadherin plays a pivotal role in tissue morphogenesis by forming clusters that support intercellular adhesion and transmit tension. What controls E-cadherin mesoscopic organization in clusters is unclear. This study used 3D superresolution quantitative microscopy in Drosophila embryos to characterize the size distribution of E-cadherin nanometric clusters. The cluster size follows power-law distributions over three orders of magnitude with exponential decay at large cluster sizes. By exploring the predictions of a general theoretical framework including cluster fusion and fission events and recycling of E-cadherin, two distinct active mechanisms setting the cluster-size distribution were identified. Dynamin-dependent endocytosis targets large clusters only, thereby imposing a cutoff size. Moreover, interactions between E-cadherin clusters and actin filaments control the fission in a size-dependent manner. It is concluded that E-cadherin clustering depends on key cortical regulators, which provide tunable and local control over E-cadherin organization. The data provide the foundation for a quantitative understanding of how E-cadherin distribution affects adhesion and might regulate force transmission in vivo (Truong Quang, 2013).
F-BAR proteins are prime candidates to regulate membrane curvature and dynamics during different developmental processes. This study analyzed nostrin (nost), a novel Drosophila F-BAR protein related to Cip4. Genetic analyses revealed a strong synergism between nost and cip4 functions. While single mutant flies are viable and fertile, combined loss of nost and cip4 results in reduced viability and fertility. Double mutant escaper flies show enhanced wing polarization defects and females exhibit strong egg chamber encapsulation defects. Live-imaging analysis suggests that the observed phenotypes are caused by an impaired E-cadherin membrane turnover. Simultaneous knock-down of Cip4 and Nostrin strongly increases the formation of tubular E-cadherin vesicles at adherens junctions. Cip4 and Nostrin localize at distinct membrane subdomains. Both proteins prefer similar membrane curvatures but seem to form different membrane coats and do not heterooligomerize. These data suggest an important synergistic function of both F-BAR proteins in membrane dynamics. A cooperative recruitment model is proposed in which first Cip4 promotes membrane invagination and early actin-based endosomal motility while Nostrin makes contact with microtubules through the kinesin Khc-73 for trafficking of recycling endosomes (Zobel, 2014).
The distribution of proteins has been analyzed in the apico-lateral cell junctions in Drosophila imaginal
discs. Antibodies to phosphotyrosine (PY),
Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous
actin, label the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin III
(FasIII) and Coracle (Cor) label the more basal septate junction. The junctional proteins labeled by
these antibodies undergo specific changes in distribution during the cell cycle. Previous work has shown that a loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to
loss of the septate junctions and the formation of what appear to be ectopic adherens junctions . This study was extended to examine the effects of mutation in other genes that
also cause imaginal disc overgrowth. Based on staining with PY and Dlg antibodies, the apico-lateral
junctional complexes appear normal in tissue from the hyperplastic overgrowth mutants fat (coding for a novel cadherin) , discs overgrown, giant discs
and warts (coding for a homolog of human myotonic dystrophy kinase). However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lethal (2) giant larvae show
abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in
loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral
junctions are required both for apico-basal cell polarity and for the signaling mechanisms controlling
cell proliferation, whereas others are required more specifically in cell-cell signaling (Woods, 1997).
In Drosophila, Src oncogene 1 was considered a unique ortholog of the vertebrate c-src; however, more recent evidence has been shown to the contrary. The
closest relative of vertebrate c-src found to date in Drosophila is not Dsrc64, but Dsrc41, a gene identified for the first time in this paper.
In contrast to Src64, overexpression of wild-type Src41 causes little or no appreciable phenotypic change in Drosophila.
Both gain-of-function and dominant-negative mutations of Src41 cause the formation of supernumerary R7-type neurons,
suppressible by one-dose reduction of boss, sevenless, Ras1, or other genes involved in the Sev pathway. Dominant-negative
mutant phenotypes are suppressed and enhanced, respectively, by increasing and decreasing the copy number of wild-type
Src41. The colocalization of Src41 protein, actin fibers and DE-cadherin, as well as the Src41-dependent disorganization of actin fibers
and putative adherens junctions in precluster cells, suggest that Src41 may be involved in the regulation of cytoskeleton
organization and cell-cell contacts in developing ommatidia (Takahashi, 1996).
Dynamically regulated cell adhesion plays an important role during animal morphogenesis. The formation of the visual system in Drosophila embryos has been used as a model system to investigate the function of the Drosophila classic cadherin, DE-cadherin, which is encoded by the shotgun (shg) gene. The visual system is derived from the optic placode, which normally invaginates from the surface ectoderm of the embryo and gives rise to two separate structures, the larval eye (Bolwig's organ) and the optic lobe. The optic placode dissociates and undergoes apoptotic cell death in the absence of Shotgun, whereas overexpression of Shotgun results in the failure of optic placode cells to invaginate and of Bolwig's organ precursors to separate from the placode. These findings indicate that dynamically regulated levels of Shotgun are essential for normal optic placode development. Overexpression of Shotgun can disrupt Wingless signaling through titration of Armadillo out of the cytoplasm to the membrane. However, the observed defects are likely the consequence of altered Shotgun mediated adhesion rather than a result of compromising Wingless signaling, since overexpression of a Shotgun-alpha-catenin fusion protein, which lacks Armadillo binding sites, causes defects similar to Shotgun overexpression. The genetic interaction between Shotgun and the Drosophila EGF receptor homolog, Egfr, was studied. If Egfr function is eliminated, optic placode defects resemble those following Shotgun overexpression, which suggests that loss of Egfr results in an increased adhesion of optic placode cells. An interaction between Egfr and Shotgun is further supported by the finding that expression of a constitutively active Egfr enhances the phenotype of a weak shg mutation, whereas a mutation in rhomboid (rho) (an activator of the Egfr ligand Spitz) partially suppresses the shg mutant phenotype. Finally, Egfr can be co-immunoprecipitated with anti-Shotgun and anti-Armadillo antibodies from embryonic protein extracts. It is proposed that Egfr signaling plays a role in morphogenesis by modulating cell adhesion (Dumstrei, 2002).
The head ectoderm of early Drosophila embryos is subdivided into several domains that realize different
morphogenetic programs. The embryonic eye field is the posterior-medial region of the procephalic neurectoderm that gives rise to the visual system, which includes the larval eye (Bolwig's organ) and adult
eye, as well as the optic lobe. Around gastrulation, cells of the eye field undergo a convergent extension directed laterally. Shortly afterwards these cells form two morphologically visible placodes, one on either side of the embryo. These optic placodes sink inside and become the optic lobe primordia, epithelial double layers attached to the posterior surface of the brain. The optic placode of a stage 12-13 embryo is V-shaped, with the anterior leg of the V representing the anterior lip, which later forms the inner anlage of the optic lobe, and the posterior leg forming the posterior lip, later forming the outer anlage. As the invagination deepens and the two lips 'sink' inside the embryo, ectodermal cells that earlier surrounded the perimeter of the optic placode approach each other and eventually form a
closed epidermal cover. Abundant cell death accompanies the closing of the head epidermis over the optic lobe anlage, and the subsequent separation of
this anlage from the epidermis. A small number of cells that originally formed part of the posterior lip of the optic placode
remain integrated in the head epidermis and form the larval eye or Bolwig's organ. As these cells move away from the optic lobe anlage their basal ends
become drawn out and form axons that constitute Bolwig's nerve (Dumstrei, 2002).
Shotgun is expressed throughout the ectoderm including the eye field and its epithelial derivatives. One would expect that normal optic
lobe development requires modulation of Shotgun activity to allow, for example, the segregation of the invaginating optic placodes from the
surrounding ectoderm. Since cell culture studies have indicated that the mammalian EGF receptor can disrupt cadherin-based adhesion, it was of interest to see whether Drosophila Egfr is expressed in the visual system to allow for such a possibility in Drosophila as well. Egfr is
expressed in a complex and dynamic pattern that closely parallels the pattern of double-phosphorylated ERK (dpERK) expression, indicating activation
of the MAP kinase signaling pathway. During stage 7 both rho (an activator of Egfr signaling) and dpERK are expressed in two stripes in the head ectoderm. The expression of dpERK in these two stripes is the result of Egfr activity. The anterior stripe corresponds to part of the head midline, while the posterior stripe reaches into the eye field. Distribution of dpERK in the two stripes becomes patchy during stage 10. At the
same time, the posterior stripe widens dorsally to overlap with part of the optic lobe placode. Finally, at the late extended germ band stage and
during germ band retraction, dpERK becomes restricted to the optic lobe placodes and cells of the dorsal head midline. This expression pattern demonstrates that Egfr activation accompanies the determination, morphogenesis and differentiation of the embryonic visual system (Dumstrei, 2002).
On the subcellular level, Egfr is expressed diffusely on the membrane of epithelial cells and neuroblasts. Egfr overlaps with Armadillo,
the Drosophila ß-catenin homolog, which is an integral component of the cadherin-catenin complex. Like Shotgun, Armadillo is
concentrated strongly in the apically located zonula adherens but is also found at lower levels in the entire lateral membranes (Dumstrei, 2002).
A second type of junction, called a septate junction, develops in Drosophila epithelial cells at a slightly later stage than the zonula adherens. Septate junctions have been implicated in maintaining epithelial stability. The Coracle protein forms part of
the septate junctional complex, and an antibody against Coracle serves as a sensitive marker for this junction. Applying this
marker to embryos of different stages it was found that all ectodermally derived epithelia express Coracle, except for the optic lobe and the invaginations
that form the stomatogastric nervous system. Accordingly, no septate junctions have been reported in previous electron microscopic
investigations of these tissues. The reliance on adherens junctions alone may make the optic lobe (and stomatogastric nervous system) susceptible to changes in the stability of these junctions; such changes occur resulting from manipulations of Shotgun and Egfr (Dumstrei, 2002).
A finely adjusted level of Shotgun is required for optic placode morphogenesis, and ß-catenin, as well as EGFR signaling, is involved in this process. Reduction in Shotgun results in dissociation of the placode around the time when it normally invaginates, suggesting that the forces exerted on the epithelial sheet while folding may disrupt cell contacts. A similar phenotype was described for other epithelial invaginations, including the Malpighian tubules and stomatogastric nervous system. Abolishing Armadillo/ß-catenin function results in a similar, if somewhat weaker phenotype. If Shotgun is overexpressed, invagination is also impaired. Cells stay together in a placode-like formation (as would be expected from 'hyperadhesive' epithelial cells), but do not noticeably constrict apically. It should be noted that the interpretation of this failure of optic placode cells to constrict is complicated by the accompanying increase in cell death in surrounding head epidermal cells. This phenomenon, in addition to a direct effect of an increased amount of Shotgun in the optic placode cells, could be part of the pathology responsible for the non-invagination phenotype. By contrast, the non-disjunction of optic lobe and larval eye is likely to be a rather direct consequence of an increased amount of Shotgun expression. Interestingly, other adhesion systems, notably the Drosophila N-CAM homolog FasII, are also involved in optic lobe-larval eye separation. Thus, the down regulation of FasII by the 'anti-adhesion' molecule Beaten path is also required for normal larval eye morphogenesis (Dumstrei, 2002).
Overexpression of Shotgun or the DE-cad-alpha-catenin fusion construct causes a dramatic change in optic lobe morphogenesis, without causing much disruption in other epithelia. It is speculated that this enhanced sensitivity of optic lobe cells towards an increased level of Shotgun may be in part due to the fact that adherens junctions form the only means of contact between optic lobe cells. In other epithelia, such as epidermis, trachea and hindgut, septate junctions form by far the more prominent junctional complex. Septate junctions have been implicated in epithelial stability. One could surmise that embryonic epithelia, as they enter the phase of differentiation during mid-embryogenesis, construct septate junctions that add to the adherens junctions developed at an earlier stage. This additional junctional complex makes late epithelia more resistant to changes in cadherins, a notion supported by the finding that blocking cadherins (by applying calcium chelators, or tyrosine kinase inhibitors) in early embryos up to stage 10 leads to a break down of epithelia, whereas it has only a small effect in later stages. The optic lobe, which does not differentiate but gives rise to a population of neuroblasts later dring the larval period, does not form septate junctions, which could account for its strong reliance on normally functioning adherens junctions (Dumstrei, 2002).
Expression of a fusion construct, DE-cad-alpha-catenin, in which the cytoplasmic domain of Shotgun is replaced by a truncated alpha-catenin, thereby preventing a reduction in the cytoplasmic pool of Arm, results in a similar phenotype as overexpressing full length Shotgun. This finding lends support to the notion that dissociation of the cadherin-catenin complex (CCC) may not occur at the interface between Shotgun and Arm or Arm and alpha-catenin. If one were to assume that dissociation occurred between any components of the CCC, one would expect a stronger phenotype, given that by overexpressing the fusion construct one not only increases the amount of Shotgun molecules interconnecting cells, but also the stability with which they are coupled to the cytoskeleton. Biochemical studies in vertebrates and Drosophila also show that phosphorylation of the CCC does not result in increased dissociation of Arm or alpha-catenin from the CCC, suggesting that the dissociation occurs distal to alpha-catenin (Dumstrei, 2002).
The strength of the CCC and other structural molecules driving morphogenesis has to be controlled in a complex spatiotemporal pattern. Numerous widely conserved signaling pathways have been implicated in this process. In vertebrate embryos, mutations of the Wnt, Shh and BMP signaling pathways result in impressive examples which tissues and organs show defects in morphogenesis. Furthermore, it became clear that frequently signaling proteins affect fundamental cellular behaviors, such as proliferation, motility, adhesiveness and survival. This prompted the hypothesis that in many developmental scenarios, the 'proximal' effect of receiving a signal could be a change in morphogenetic behavior. The discovery that one of the Wnt signal transducers, ß-catenin, leads a 'double life' as a structural component of the cadherin-catenin complex, fueled the idea that Wnt signal could directly exert an effect on the adhesiveness on the cell, an idea that is supported by cell culture experiments. However, genetic studies have demonstrated that in Drosophila, the roles of ß-catenin as a signaling transducer and a CCC component seem to be quite separate. Although it is clear that the cytoplasmic and membrane bound ß-catenin pools are in a steady state, binding of more ß-catenin to the membrane, by overexpression of Shotgun, reduces the cytoplasmic pool resulting in a wg minus phenotype. However, Wnt/Wg signaling seems to have no effect on the amount of membrane bound ß-catenin. Thus, in Drosophila, it appears that Shotgun mediated adhesion, at least under experimental conditions, interferes with Wnt/Wg signaling by competing for ß-catenin but Wnt/Wg signaling may not have a direct effect on adhesion mediated by the CCC (Dumstrei, 2002).
The findings suggest that another signaling pathway, the Egfr pathway, is involved in modulating cadherin-mediated adhesion and thereby controls morphogenesis. Egfr, similar to its function in the developing compound eye, is activated in the precursors of the larval eye and adjacent optic lobe at a stage preceding optic lobe invagination and larval eye separation. The ligand for Egfr is Spitz, which is activated by Rhomboid (Dumstrei, 2002).
In a small subset of larval eye precursors (the 'Bolwig's organ founders') loss of Egfr signaling results primarily in cell death, lending further support to the view that Egfr signaling functions generally in the ectoderm and its derivatives to maintain cell viability. Recent studies in Drosophila indicate that MAPK directly controls the expression and protein stability of the cell death regulator, Hid (W; Wrinkled). If cell death is prohibited by a deficiency of the reaper-complex, cells of the optic placode and most other embryonic cells that undergo apoptosis in Egfr loss-of-function mutants survive. Both optic lobe and Bolwig's organ express several of their normal differentiation markers, but show a characteristic 'hyperadhesive phenotype', consisting in the failure of optic Iobe invagination and Bolwig's organ separation. Based on the similarity of this phenotype to the one resulting from Shotgun overexpression, and the genetic interaction between Egfr and Shotgun mutants in the ventral ectoderm, it is proposed that Egfr activation is required in normal development to phosphorylate the CCC and thereby allows optic lobe invagination and Bolwig's organ separation to occur. This would be in line with experimental results obtained in vertebrate cell culture studies, which have demonstrated that drug- or Egfr-induced phosphorylation of the CCC leads to dissociation between CCC and cytoskeleton. Recent findings have shown that another phosphorylation event, mediated by the rho/rac GTPases, also affects adhesion by dissociating alpha-catenin from the CCC (Dumstrei, 2002).
Co-IP data indicates that Egfr is linked to the CCC in Drosophila as well. This implies that the effect of Egfr on Shotgun mediated adhesion could be a direct one that occurs at the cell membrane and does not involve MAPK signal transduction to the nucleus. It has been shown in a number of vertebrate cell culture systems that tyrosine phosphorylation of ß-catenin results in a disassembly of the CCC complex and a consecutive loss in cadherin-mediated adhesion. Phenotypically, this results in increased invasiveness of tumor cell lines, neuronal and growth cone motility. Several tyrosine kinases and phosphatases have been identified that can increase or decrease the degree of phosphorylation of the CCC. For example, v-src transfected into cultured cells phosphorylates ß-catenin and causes cells to dissociate, round up, and become more motile. Egfr also phosphorylates the CCC and forms an integral part of this complex. This opens up the possibility that growth factors, with their widespread expression and biological activity in the developing embryo, may exert part of their effect on cell behavior by modulating, in a rather direct way, cell adhesion at the membrane. Such a mechanism would account for the 'rapid mode' of control of adhesion molecules. Systems such as the optic placode of the Drosophila embryo, where matters of different cell fates are decided at the same time when morphogenetic movements change the arrangement and shape of the cells involved, constitute favorable paradigms to address how signaling systems control both processes (Dumstrei, 2002).
Epithelial remodelling is an essential mechanism for organogenesis, during which cells change shape and position while maintaining contact with each other. Adherens junctions (AJs) mediate stable intercellular cohesion but must be actively reorganised to allow morphogenesis. Vesicle trafficking and the microtubule (MT) cytoskeleton contribute to regulating AJs but their interrelationship remains elusive. This study reports a detailed analysis of the role of MTs in cell remodelling during formation of the tracheal system in the Drosophila embryo. Induction of MT depolymerisation specifically in tracheal cells showed that MTs were essential during a specific time frame of tracheal cell elongation while the branch extended. In the absence of MTs, one tracheal cell
per branch overelongated, ultimately leading to branch break.
Three-dimensional quantifications revealed that MTs were crucial to
sustain E-Cadherin (Shotgun) and
Par-3 (Bazooka) levels at AJs.
Maintaining E-Cadherin/Par-3 levels at the apical domain required de
novo synthesis rather than internalisation and recycling from and to
the apical plasma membrane. However, apical targeting of E-Cadherin
and Par-3 required functional recycling endosomes, suggesting an
intermediate role for this compartment in targeting de novo
synthesized E-Cadherin to the plasma membrane. The apical enrichment
of recycling endosomes was dependent on the MT motor Dynein and essential for the
function of this vesicular compartment. In addition, E-Cadherin
dynamics and MT requirement differed in remodelling tracheal cells versus planar epithelial cells. Altogether, these results uncover an MT-Dynein-dependent apical restriction of recycling endosomes that controls adhesion by sustaining Par-3 and E-Cadherin levels at AJs during morphogenesis (Le Droguen, 2015).
This study has reveal the importance of the functional interplay between MTs, vesicular trafficking and the control of AJ dynamics in cells undergoing extensive remodelling through collective migration. MT depletion in tracheal cells induces the formation of intracellular dots containing E-Cad and Par-3. Interestingly, these intracellular accumulations are only seen in remodelling tracheal cells and not in planar epithelia. These dots are still detected when endocytosis is affected, showing that they are de novo synthesis route intermediates. Altogether, this suggests that maintaining the correct level of E-Cad/Par-3 at the apical domain requires a continuous supply of newly synthesized proteins, which could be essential for the intensive AJ reorganisation that occurs during cell intercalation and elongation of the tracheal branch (Le Droguen, 2015).
Using the photo-convertible E-Cad-EosFP in flat epithelium, a previous study showed that E-Cad that is engaged in homophilic interactions at the AJs forms very stable domains. This study has demonstratde that MT depletion does not affect the integrity of this 2D epithelium. In addition, it was shown that the pool of photo-converted E-Cad-EosFP is less stable in tracheal cells than in epidermal cells. Together with a FRAP assay suggesting that AJs are more dynamic in tracheal cells than in epithelial cells, the results highlight a specific fine-tuning of AJ components in tracheal cells undergoing cell movement and cell shape changes in 3D through cell intercalation, cell elongation and thereby organ formation. This fine-tuning is likely to cycle between internalisation, recycling, degradation and de novo synthesis, the latter being MT dependent. When the balance is altered in the absence of MTs, and thus when E-Cad or Par-3 are reduced at AJs, tracheal cells overelongate after completing intercalation. During branch elongation without MTs, the two migrating leading cells generate a pulling force on the following stalk cells, which display a critical reduction in AJ components. Either the tip cell or the base cell of the stalk becomes unable to maintain its integrity in response to this force. Consequently, this tracheal cell overelongates by a factor 1.8, preventing the remaining stalk cells from reaching their average size. As a result, DBs present a single overelongated cell and several underelongated cells (Le Droguen, 2015).
An overlap was detected between the E-Cad intracellular dots generated in the absence of MTs and recycling endosome vesicles. Interfering with Rab11 function in tracheal cells induces cell overelongation and affects E-Cad and Par-3 distribution at the AJs, as does MT depletion. These results illustrate that the E-Cad de novo synthesis pathway passes through the Rab11-positive recycling endosome compartment. The overlap of E-Cad and recycling endosome markers represents only a small proportion of the total intracellular E-Cad, suggesting a transient residence in this vesicular compartment for this newly synthesized protein. Recent studies conducted in different model systems have revealed that some newly synthesized apical plasma membrane proteins, such as E-Cad and Rhodopsin, leave the trans-Golgi network to cross Rab11-positive recycling endosome compartments before reaching the apical surface). This apical trafficking route is used specifically in tracheal cells and requires the MT network. Quantification of Rab11DN-associated defects upon tracheal branch formation reveals that impairing recycling endosome function has a similar effect to altering the distribution of E-Cad at AJs by depleting the MT network. Moreover, interfering with Rab11 function reduces Par-3 levels at the AJs of tracheal cells. Interestingly, Par-3 does not colocalise with the E-Cad cytoplasmic pool, indicating that functional recycling endosomes are required by Par-3 and E-Cad to assemble as a complex and to be targeted to the apical domain of tracheal cells. However, as E-Cad and Par-3 can be apically targeted in the absence of the other, this suggests that apical targeting of E-Cad and Par-3 can be independent in tracheal cells or that redundant pathways could sustain the localisation of each protein. For example, the Nectin protein Echinoid is required for Par-3 localisation at AJs in shg mutant cells. Moreover, the apical distribution of PI(4,5)P2 in the Drosophila follicular epithelium sustains Par-3 apical anchorage at the plasma membrane. Furthermore, Par-3-independent localisation of E-Cad has been observed (Le Droguen, 2015).
Dynamic MTs in the ectoderm locally upregulate AJ turnover through RhoA activity. RhoA stabilises cellular contacts through acto-myosin regulation. In tracheal cells, MT depletion does not alter actin distribution. Moreover, tracheal cells mutant for the Myosin light chain zipper (zip) and also expressing a dominant-negative form of Zip do not present obvious defects in E-Cad distribution at stage 14. By contrast, MT depletion induces the cytoplasmic accumulation of E-Cad and Par-3 in tracheal cells only and not in ectodermal cells at the same developmental stage. Thus far, cytoplasmic accumulation of E-Cad and Par-3 has only been observed after colchicine-induced MT depolymerisation during polarity establishment at embryo cellularisation, when AJs are extremely dynamic and vesicular trafficking is strongly active. This study demonstrated that the E-Cad distribution in tracheal cells is more sensitive to MT depolymerisation and to Rab11DN overexpression than that in the overlying ectodermal cells. The comparison of the maximum recovery of the E-Cad signal in a FRAP assay in tracheal cells and in ectodermal cells, together with the differences in stability of the photo-converted E-Cad-EosFP between these two tissues, suggest that AJs are more dynamic in tracheae. It is thus conceivable that tracheal cells, which undergo extensive cell shape changes through collective cell migration, require more dynamic AJs as sustained by an efficient targeting of E-Cad. These discrepancies between ectodermal and tracheal epithelia underline the importance of investigating MT function in different morphogenetic contexts under different constraints (Le Droguen, 2015).
This study has demonstrated that the MT minus-end motor Dynein is essential for the restricted localisation of recycling endosomes in a developing organism. The Dynein requirement for the apical enrichment of recycling endosomes is in agreement with the MT minus ends being anchored at the apical plasma membrane. The asymmetric distribution of recycling endosome vesicles has been observed in various differentiated cell types, especially during cell division. Vesicles are found enriched either in the apical domain or at the microtubule-organising centre (MTOC; i.e. the centrosome) during mitosis. Indeed, in vivo, Dynein physically interacts with Nuf. During metaphase, Dynein is required for the maintenance of Nuf at the centrosome. This study demonstrates that Dynein is also required for the apical distribution of recycling endosomes in non-dividing tracheal cells. In a context in which Dynein function is altered, Nuf-positive recycling endosome vesicles are dispersed but colocalise with E-Cad and Par-3 intracellular dots, indicating that the recycling endosome compartment remains functional for the assembly of such a complex (Le Droguen, 2015).
A previous study has characterised the relocalisation of the MTOC in tracheal cells; MTs are nucleated and anchored at the apical domain just above the AJs. It has also been demonstrated that such MT organisation is crucial for tracheal morphogenesis. Non-centrosomal MT organisation occurs in many differentiated cell types but the functional relevance of such an organisation is still poorly understood. It will be informative to investigate whether such non-centrosomal MT organisation provides a means to regulate epithelial remodelling by controlling the apical enrichment of recycling endosomes and thus AJ dynamics (Le Droguen, 2015).
The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in
both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and
mammals have two APC family members. The functions of the Drosophila APCs is further explored using the larval brain
as a model. Both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions.
Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and
APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize
to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and
microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. These experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins (Akong, 2002).
One striking feature of the asymmetric localization of
APC2 is that it is present throughout the cell cycle and is
particularly strong during interphase. During embryonic
neuroblast divisions, most asymmetric markers are localized only
during mitosis. However, less is known about their localization in larval
neuroblasts. Several asymmetric markers
in larval neuroblasts were examined, and
their localization was compared with that of APC2. In embryonic
neuroblasts, the transcription factor Prospero (Pros)
and its mRNA are GMC determinants that are asymmetrically
localized to the GMC daughter. Pros protein then becomes nuclear and helps
direct cell fate. In larval neuroblasts, a similar localization is observed. Pros is not detectable in interphase neuroblasts, when the cortical APC2 crescent is strongest. A small amount of Pros transiently localizes to an asymmetric crescent during mitosis. Pros is present at low levels in GMC nuclei and at higher levels in the nuclei of ganglion cells (Akong, 2002).
Mira is basally localized in embryonic neuroblasts,
and required there for localization of Pros protein
and mRNA. In central brain neuroblasts, Mira is diffusely cytoplasmic
during interphase, when the APC2 crescent is the
strongest. As cells enter mitosis, Mira first becomes cortical and then begins to accumulate asymmetrically on
the side of the neuroblast where the daughter will be born. By metaphase, Mira asymmetry is very pronounced. The center of the Mira crescent is always precisely aligned with one spindle pole. As a result, in cells with the
spindle pointing toward the center of the APC2 crescent,
the Mira and APC2 crescents substantially overlap, while in cells in which the spindle points to the edge of the APC2 crescent, the two crescents are
offset. Mira is partitioned into the GMC during anaphase, while APC2 relocalizes to the cleavage furrow. Mira could still be detected in some GMCs, which are thought to be those that were recently born (Akong, 2002).
In contrast to Mira and Pros, Inscuteable (Insc) and
Bazooka (Baz) localize to the apical sides of embryonic
neuroblasts, where they play essential roles in asymmetric
divisions. Insc is asymmetrically localized in larval neuroblasts. Insc localizes to the side of the neuroblast opposite that of APC2 through much, if not all, of the cell cycle. Interestingly, there is a weak Insc crescent during interphase, that becomes stronger through prophase
and metaphase. During anaphase, Insc
localizes to the neuroblast cortex but not the GMC daughter. Baz localization was similar to that of Insc,
though no cortical localization during interphase was detected. During prophase and metaphase, Baz
localizes to a crescent opposite APC2, and as
the chromosomes begin to separate, Baz localizes to a tight
cap opposite the future GMC. Together, these data
confirm that larval and embryonic neuroblasts asymmetrically
localize many of the same proteins, and that APC2
localizes on the GMC side (basal) of the neuroblast, overlapping
Mira and opposite Baz and Insc, which localize apically (Akong, 2002).
Arm also localizes asymmetrically in neuroblasts. Extending this, an examination was made of the localization of Arm's adherens junction partners DE-cadherin and ß-catenin. When central brain neuroblasts undergo a sequential
series of asymmetric divisions, the GMCs remain
associated with their neuroblast mother, resulting in a cap
of GMCs in association with each neuroblast. APC2 localizes
strongly to the boundary between the neuroblast and
each GMC, and more weakly to the borders between the GMCs. APC2 is present at lower levels in ganglion cells and differentiating neurons (Akong, 2002).
The adherens junction proteins DE-cadherin, Arm, and
ß-catenin all show a striking and asymmetric localization
pattern in central brain neuroblasts. All
precisely colocalize both at the boundary between neuroblasts
and GMCs and at the boundaries between GMCs. DE-cadherin, Arm, and
ß-catenin are also all expressed in epithelial cells of the
outer proliferation center. The localization of DE-cadherin and the catenins is consistent with the idea that cadherin-catenin-based adhesion
could help ensure that GMCs remain associated with
each other, via association with their neuroblast mother (Akong, 2002).
To further explore this, how successive
GMCs are positioned relative to their older GMC sisters was examined
using two different approaches. First Mira was used to mark the newborn GMCs and DE-cadherin was used to mark the neuroblast and all of her GMC
daughters. Mira localizes to a crescent on the side of the
neuroblast where the daughter will be born (basal side), and then is
segregated into the daughter. Mira persists for some time in newborn GMCs, and it remains detectable in the other GMCs as well, thus
allowing the position of newborn GMCs to be examined relative to their older sisters. In many cases, new GMCs are clearly born at the edge of the cluster of older GMCs. This is particularly striking in neuroblasts with many progeny. It is worth noting that the cluster of daughters is three-dimensional, comprising a 'cap' of daughters in three dimensions rather than a two-dimensional line of daughters. It is thus suspected that new daughters are born near the edge of this cap (Akong, 2002).
These data suggest that neuroblasts and their GMC
progeny remain closely associated. The GMCs then divide
to form ganglion cells and ultimately neurons. The data
further suggest that these latter cells may also remain
associated and send their axons together toward targets in
the central brain. When sections were made more deeply into the
brain, below each cluster of neuroblasts and GMCs,
structures that appear to be axons were detected projecting from these
groups of cells. These axons label with Arm, DE-cadherin, and APC1. Arm also localizes to the axons of the neuropil, while DE-cadherin and APC2 are present at low levels or are absent from this structure (Akong, 2002).
Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). A new morphogenetic/cellular mechanism for disc eversion has been uncovered. Imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. The JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells (Pastor-Pareja, 2004).
One hallmark of epithelial cells is their distinct apico-basal cell polarity. This polarity depends on a set of intercellular connections, which encircle epithelial cells at the border of the apical and basal-lateral membrane domains. The cells in insect epithelial tissues are interconnected by zonula adherens (ZAs), which function in both cellular adhesion and signaling. DE-cadherin is the major constituent of the ZAs in a complex with Armadillo (Arm, ß-catenin) and Dalpha-catenin. In addition, epithelia of flies and other invertebrates exhibit septate junctions, which are located basally to the ZAs. Septate junctions prevent diffusion through the pericellular space and are functionally equivalent to vertebrate tight junctions (Pastor-Pareja, 2004).
All imaginal disc cells at the third instar larval stage presented ZAs in an apical belt. During disc eversion, however, it was found that ZAs components delocalize from the free edges of the PS cells, remaining cytoplasmic at the edges of the perforations arising through the PS/larval bilayer and in those PS cells leading the spreading of the discs over the larval tissues. As a consequence, ZAs are lost in these cells. Moreover, septate junction components, such as Coracle and Disc Large are also found to be missing from the membranes of leading front cells (Pastor-Pareja, 2004).
The loss of apico/basal polarity and adhesion of the PS cells during disc eversion is reminiscent of an epithelial-mesenchymal transition (EMT), as described for mesoderm and neural crest cells in vertebrates, and for the acquisition of the invasive phenotype in carcinomas (Pastor-Pareja, 2004).
In summary, the evagination of imaginal disc can be divided into the following sequential steps: (1) an overall positional change of the imaginal discs leading to the confrontation and apposition of the PS and the larval epidermis; (2) a regulated modulation (PEMT) of PS cells, which involves the downregulation of their cell-cell adhesion systems and allows them to move into their local neighborhood and invade the larval epithelium; (3) the fenestration of the peripodial/larval bilayer and the formation of an unbound peripodial leading front, which will direct imaginal spreading by planar cell intercalation, and (4) a bulging of the imaginal tissue (Pastor-Pareja, 2004).
Once the hole is opened, the planar intercalation of PS cells ensures that, first in the hole and later in the leading front, all four dorsal, ventral, anterior, and posterior compartments of the wing disc are represented. This mechanism also guarantees the maintenance of a continuous epithelial barrier (Pastor-Pareja, 2004).
Armadillo, the Drosophila homolog of β-catenin, plays a crucial role in both the Wingless signal transduction pathway and cadherin-mediated cell-cell adhesion, raising the possibility that Wg signaling affects cell adhesion. This study used a tissue culture system that allows conditional activation of the Wingless signaling pathway and modulation of E-cadherin expression levels. Activation of the Wingless signaling pathway leads to the accumulation of hypophosphorylated Armadillo in the cytoplasm and in cellular processes, and to a concomitant reduction of membrane-associated Armadillo. Activation of the Wingless pathway causes a loss of E-cadherin from the cell surface, reduced cell adhesion and increased spreading of the cells on the substratum. After the initial loss of E-cadherin from the cell surface, E-cadherin gene expression is increased by Wingless. It is suggested that Wingless signaling causes changes in Armadillo levels and subcellular localization that result in a transient reduction of cadherin-mediated cell adhesion, thus facilitating cell shape changes, division and movement of cells in epithelial tissues (Wodarz, 2006; full text of article).
The transcription factors TBX2 and TBX3 are overexpressed in various human cancers. This study investigated the effect of overexpressing the orthologous Tbx genes Drosophila optomotor-blind (omb) and human TBX2 in the epithelium of the Drosophila wing imaginal disc; two types of cell motility were observed. Omb/TBX2 overexpressing cells could move within the plane of the epithelium. Invasive cells migrated long-distance as single cells retaining or regaining normal cell shape and apico-basal polarity in spite of attenuated apical DE-cadherin concentration. Inappropriate levels of DE-cadherin were sufficient to drive cell migration in the wing disc epithelium. Omb/TBX2 overexpression and reduced DE-cadherin-dependent adhesion caused the formation of actin-rich lateral cell protrusions. Omb/TBX2 overexpressing cells could also delaminate basally, penetrating the basal lamina, however, without degradation of extracellular matrix. Expression of Timp, an inhibitor of matrix metalloproteases, blocked neither intraepithelial motility nor basal extrusion. These results reveal an MMP-independent mechanism of cell invasion and suggest a conserved role of Tbx2-related proteins in cell invasion and metastasis-related processes (Shen, 2014).
When exposed to DNA-damaging agents, components of the DNA damage response (DDR) pathway trigger apoptosis, cell cycle arrest and DNA repair. Although failures in this pathway are associated with cancer development, the tumor suppressor roles of cell cycle arrest and apoptosis have recently been questioned in mouse models. Using Drosophila epithelial cells that are unable to activate the apoptotic program, evidence is provided that ionizing radiation (IR)-induced DNA damage elicits a tumorigenic behavior in terms of E-cadherin delocalization, cell delamination, basement membrane degradation and neoplasic overgrowth. The tumorigenic response of the tissue to IR is enhanced by depletion of Okra/DmRAD54 or spnA/DmRAD51-genes required for homologous recombination (HR) repair of DNA double-strand breaks in G2-and it is independent of the activity of Lig4, a ligase required for nonhomologous end-joining repair in G1. Remarkably, depletion of Grapes/DmChk1 or Mei-41/dATR-genes affecting DNA damage-induces cell cycle arrest in G2-compromises DNA repair and enhances the tumorigenic response of the tissue to IR. On the contrary, DDR-independent lengthening of G2 has a positive impact on the dynamics of DNA repair and suppressed the tumorigenic response of the tissue to IR. These results support a tumor suppressor roles of apoptosis, DNA repair by HR and cell cycle arrest in G2 in simple epithelia subject to IR-induced DNA damage (Dekanty, 2014).
Ommatidial rotation is a cell motility read-out of planar cell polarity (PCP) signaling in the Drosophila eye. Although the signaling aspects of PCP establishment are beginning to be unraveled, the mechanistic aspects of the associated ommatidial rotation process remain unknown. This study demonstrates that the Drosophila DE- and DN-cadherins have opposing effects on rotation. DE-cadherin promotes rotation; DE-cad mutant ommatidia rotate less than wild type or not at all. By contrast, the two DN-cadherins act to restrict this movement, with ommatidia rotating too fast in the mutants. The opposing effects of DE- and DN-cadherins result in a coordinated cellular movement, enabling ommatidia of the same stage to rotate simultaneously. Genetic interactions, phenotypic analysis and localization studies indicate that EGF-receptor and Frizzled-PCP signaling feed into the regulation of cadherin activity and localization in this context. Thus, DE- and DN-cadherins integrate inputs from at least two signaling pathways, resulting in a coordinated cell movement (Mirkovic, 2006).
Although the role for DE-cad in tissues undergoing rearrangements during
development is established, a direct role for DE-cad in cell and tissue movement has been more difficult to study in vivo owing to its essential role in
maintenance of epithelial integrity. Analysis of adult eye phenotypes of a
homozygous viable shg/DE-cad allele and a dominant-negative DE-cad
construct (DE-cadDN), expressed in the R3/R4 and later R1/R6, R7
precursors, indicate that DE-cad is required throughout the rotation process.
The ability of ommatidia to complete the precise 90° rotation directly
depends on DE-cad activity. Both the extracellular domain, responsible for
cell-cell adhesion, and the intracellular domain, linking DE-cad to the actin
cytoskeleton, are required for rotation. DE-cad associates with the actin
cytoskeleton primarily through interactions with Arm/ß-catenin. Although
ß-catenin has a dual role in cell adhesion and Wg signaling (which can be
separated), these data indicate that during ommatidial rotation
ß-catenin acts through its role in cell adhesion (Mirkovic, 2006).
Ommatidial rotation represents the final step in establishing PCP during
eye development. The direction of rotation depends on proper R3/R4 cell fate
specification, which is determined by PCP signaling. The Egfr pathway and
input by rotation-specific genes, e.g. nemo, are thought to function
in parallel to Fz-PCP signaling. An enhancement of the
sev>DE-cadDN rotation defects was observed by dose reduction in core regulatory PCP genes dgo and stbm; ommatidial under-rotation and the number of ommatidia that did not initiate rotation in
sev>DE-cadDN/dgo-/+, stbm-/+ was
comparable with the enhancement of sev>DE-cadDN by heterozygosity
of a shg null allele). The localization of PCP protein complexes at
the level of adherens junctions is consistent with the idea that PCP factors can influence DE-cad function. The mechanism of this regulation remains unclear. The
RhoA-RNAi transgene, which was expressed only in R3/R4 precursors
during the initiation of ommatidial rotation, enhanced
sev>DE-cadDN associated under-rotation defects. Although a RhoA
requirement in multiple cellular processes makes it difficult to dissect its
specific role in rotation, the specificity of the phenotype (enhanced
under-rotation in sev>DEcad/RhoAIR) suggests a role for
RhoA in the regulation of cadherin-mediated cell movement (Mirkovic, 2006).
Although Egfr signaling appears to be required for the precise 90°
rotation, its role in the process - promoting motility or antagonizing
it - has remained unclear. The genetic data suggest that Egfr signaling acts
positively to promote rotation, since a reduction in Egfr signaling enhances the
sev>DE-cadDN under-rotation phenotype. This may reflect a
positive role for Egfr signaling in the regulation of DE-cad activity or
turnover at the membrane, as suggested from human tumor cell lines. Affecting
the function of endocytic pathway components can also have an effect on
ommatidial rotation. This might be mediated by Egfr signaling, as is thought to
be the case in human cancer cells, leading to recycling and redistribution of
E-cad at the plasma membrane (Mirkovic, 2006).
Drosophila DN-cadherins, which are encoded by the adjacent
cadN and cadN2 genes, are the main cadherins expressed in
the nervous system. In developing photoreceptors they participate in axon
guidance, and in pupal eye discs they mediate terminal patterning of the
retina [through specific expression in cone cells. During
PCP establishment, DN-cad1 is concentrated at the border between R3/R4
precursors, in a pattern largely complementary to DE-cad. This suggested a
possible combinatorial role for DE-cad and DN-cad in rotation, with DN-cad
either providing a structural role in rotating clusters, or participating in
signaling cascades that regulate cell movement. Analysis of DN-cad
mutant clones in discs during rotation demonstrated a specific function;
many mutant clusters have completed rotation well before wild-type clusters of
the same stage. These data indicate that DN-cadherins function to
slow down rotation, serving an opposing function to DE-cad (Mirkovic, 2006).
The balance and complementary distribution of DE-cad and DN-cad appear
crucial for correct rotation to occur. Mild overexpression of DN-cad1 in R3/R4
(sev>DN-cad) is sufficient to interfere with the process,
possibly by affecting DE-cad levels. Consistently, DN-cad1 overexpression
enhances sev>DE-cadDN induced under-rotation and overexpression
clones of DN-cad1 cause a decrease in endogenous DE-cad levels. Alternatively, the negative effect of DN-cad on DE-cad might be through competition for
ß-catenin, since sev>DE-cadDN is partially rescued by
UAS-ArmS2, although since
sev>DN-cad is not enhanced by arm dose reduction this
appears less likely. Interestingly, sev>DN-cad is enhanced by
co-expression of full-length DE-cad and full-length Arm. These phenotypes
resemble those of a strong sev>DN-cad line, suggesting
that DN-cad is stabilized by increased levels of available Arm, and also that
co-overexpression of two cadherins may interfere with optimal turnover rate at
the membrane (Mirkovic, 2006).
In the Drosophila retina, photoreceptor differentiation is preceded by significant cell shape rearrangements within and immediately behind the morphogenetic furrow. Groups of cells become clustered into arcs and rosettes in the plane of the epithelium, from which the neurons subsequently emerge. These cell clusters also have differential adhesive properties: adherens junction components are upregulated relative to surrounding cells. Little is known about how these morphological changes are orchestrated and what their relevance is for subsequent neuronal differentiation. This study reports that the transcription factor Atonal and the canonical EGF receptor signalling cascade are both required for this clustering and for the accompanying changes in cellular adhesion. In the absence of either component, no arcs are formed behind the furrow, and all cells show low Armadillo and DE-cadherin levels, although in the case of EGFR pathway mutants, single, presumptive R8 cells with high levels of adherens junction components can be seen. Atonal regulates DE-cadherin transcriptionally, whereas the EGFR pathway, acting through the transcription factor Pointed, exerts its effects on adherens junctions indirectly, at a post-transcriptional level. These observations define a new function for EGFR signalling in eye development and illustrate a mechanism for the control of epithelial morphology by developmental signals (Brown, 2006).
The discovery that EGFR signalling regulates cellular morphology in the morphogenetic furrow adds to the multiple functions already ascribed to this pathway in the eye. The process of cluster formation is the earliest detectable stage of ommatidial development and is tightly coordinated with subsequent photoreceptor recruitment. However, the results presented in this study clearly demonstrate that clustering is a separable process from photoreceptor differentiation. Most directly, the fact that spitz mutant clones do not show defects in clustering, whereas they fail to differentiate any photoreceptors beyond the founding R8 cell, shows that the functions of the EGFR pathway in clustering and in recruitment are distinct. In addition, these roles are spatially and temporally separable. The initial source of the Spitz signal for photoreceptor recruitment is the R8 cell. However, at the time at which the first MAPK activation is seen, in the furrow, the R8 does not yet exist. atonal expressing cells in the furrow upregulate rhomboid levels, and presumably it is these cells that release the activating ligand to control clustering. Since spitz clones do not show aberrant clustering, it is believed that Keren may be the ligand required to activate the EGFR in this process (either alone or redundantly with Spitz), since it has been conjectured that Keren is also involved in the control of ommatidial spacing, cell survival behind the morphogenetic furrow and ommatidial rotation. Evidence is available supporting the role of Keren in several aspects of eye development (Brown, 2006).
There are clearly strong similarities between the function for the EGFR described in this study and its function in the control of ommatidial spacing. In both cases, the pathway is activated in the morphogenetic furrow, under the control of Atonal, and in both, it appears as though Keren, rather than Spitz, may be the principal activating ligand. Indeed, it seems likely that the same signalling event may be responsible for coordinating both these processes. In the case of R8 spacing, it has been hypothesised that activation of the EGFR pathway leads to the secretion of an as yet unidentified inhibitory molecule, which acts non-autonomously to repress atonal expression between proneural clusters. In clustering, the signalling pathway seems to be required for two related purposes: firstly, to regulate the cell shape changes that accompany rosette and arc formation, and secondly, to maintain high levels of AJ proteins in these cells. In contrast to R8 spacing, this function appears to be cell-autonomous—no significant rescue is seen of clustering close to the borders of mutant clones. It is proposed that the same EGFR signalling event that results in the expression and secretion of the spacing inhibitory factor also causes an autonomous change in the transcriptional program of cells and leads to their maintaining strong AJs and to undergoing the cell shape changes that are required for rosette and arc formation (Brown, 2006).
Although the results demonstrate that both Atonal and the EGFR signalling cascade act to control the adhesive and morphological changes of cells behind the morphogenetic furrow, they implicate two independent mechanisms by which they act. Atonal exerts a transcriptional effect upon DE-cadherin. On the contrary, shg-lacZ and arm-lacZ levels are unaffected in ras mutant clones, indicating a function for the pathway in controlling either translation of these components, or in regulating the subcellular distribution or stability of AJs. However, it should be noted that, while the effects upon adhesion proteins are post-transcriptional, the EGFR pathway is acting through its canonical pathway and via the transcription factor Pointed, which must therefore control the expression of some downstream factor (Brown, 2006).
The following course of events is proposed. As cells enter the morphogenetic furrow, they all upregulate their levels of Armadillo and DE-cadherin. This change in the adhesive properties of the cells is independent of either Atonal or the EGFR signalling pathway, and presumably occurs as a result of the earlier signals responsible for driving the progression of the furrow, such as Hedgehog or Dpp. Since shg-lacZ expression does not appear to be upregulated at this early stage, it is speculated that this increase in protein levels is the result of some post-transcriptional mechanism, for example by stabilisation of AJs, although this has not been investigated this further. Behind the furrow, cells not fated to differentiate as photoreceptors downregulate the levels of AJ components to a low basal level. However, in the rosettes and arcs, high levels of Armadillo and DE-cadherin are maintained. This, it is suggested, is due to transcriptional upregulation of shotgun by Atonal, which is expressed in clusters of cells within the furrow before the R8 is selected, leading to the initial, broad stripe of shg-lacZ. atonal expression is then refined to the R8 precursor and presumably continues to exert its transcriptional effect on DE-cadherin in this cell, which shows the highest levels of AJ proteins. However, transcriptional upregulation of DE-cadherin cannot fully account for the maintenance of adhesion in cluster cells since over-expression of Atonal is unable to compensate for the loss of EGFR pathway activity. The results demonstrate a necessary role for the EGFR pathway in the post-transcriptional regulation of adherens junctions—presumably by promoting translation or stabilising junctional complexes to maintain strong cell–cell adhesion. Thus, these two mechanisms in concert act to promote adhesion between cells of the cluster, which are fated to form photoreceptors, while surrounding cells, which will go on to divide again, become less tightly connected (Brown, 2006).
One question that arises from the current work is the extent to which the changes in adhesive properties and the cell shape changes accompanying cluster formation are interdependent. Are the high levels of AJ proteins between cells of the cluster sufficient to reorganise the cells into distinct rosettes and arcs, or are there other mechanisms involved? Recent work has demonstrated that, later in eye development, the morphology of cone cell clusters can be accounted for solely by homophilic cadherin interactions between them; it is possible that a similar process may be occurring at this early stage (Brown, 2006).
In addition to orchestrating the cell shape changes that precede neuronal differentiation, modulation of adhesion might also be important for regulating the actual process of ommatidial cell recruitment. The observation that reduction in DE-cadherin enhances the recruitment defects caused by reduced EGFR signalling in Star mutants is consistent with a model where proper levels of DE-cadherin-mediated adhesion are required for efficient EGFR signalling. A number of previous reports are consistent with this idea. Firstly, it has been shown, both in tissue culture and in Drosophila embryos, that the EGFR co-immunoprecipitates with DE-cadherin, suggesting that cadherin might modulate EGFR activity, either directly or simply by regulating its localisation. Secondly, in mammalian tissue culture experiments, AJ formation has been shown to be capable of inducing EGFR dependent MAPK activation in a ligand independent manner. Further investigation will be required to determine details of the relationship between DE-cadherin and the EGFR, but it is interesting to consider that not only might cell signalling regulate adhesion, but that adhesion may also feed back to modulate signalling (Brown, 2006).
Correct cellular patterning is central to tissue morphogenesis, but the role of epithelial junctions in this process is not well-understood. The Drosophila pupal eye provides a sensitive and accessible model for testing the role of junction-associated proteins in cells that undergo dynamic and coordinated movements during development. Mutations in polychaetoid (pyd), the Drosophila homologue of Zonula Occludens-1, are characterized by two phenotypes visible in the adult fly: increased sensory bristle number and the formation of a rough eye produced by poorly arranged ommatidia. It was found that Pyd is localized to the adherens junction in cells of the developing pupal retina. Reducing Pyd function in the pupal eye results in mis-patterning of the interommatidial cells and a failure to consistently switch cone cell contacts from an anterior-posterior to an equatorial-polar orientation. Levels of Roughest, DE-Cadherin and several other adherens junction-associated proteins are increased at the membrane when Pyd protein is reduced. Further, both over-expression and mutations in several junction-associated proteins greatly enhances the patterning defects caused by reduction of Pyd. These results suggest that Pyd modulates adherens junction strength and Roughest-mediated preferential cell adhesion (Seppa, 2008).
The data demonstrate that Pyd is an AJ-associated protein that is required for patterning of the pupal lattice cells. Live imaging of the developing eye indicates that Pyd is necessary for the directed movements of interommatidial precursor cells (IPCs) that allow cell sorting into defined niches. Membrane contacts are dynamically exchanged in the pupal eye: each shift in the position of a cell requires the removal of previous contacts and the establishment of new ones. Pyd regulates patterning at least in part through modulating levels of the AJ-associated proteins DE-Cadherin, β-Catenin, and α-Catenin. Other studies have suggested that cell adhesion is necessary both to facilitate and restrict cell movement within the eye epithelium; the interplay between these two processes requires tight regulation of the levels of both cell adhesion molecules and junctional proteins. The data indicate that removal of Pyd from the AJ compromises this tightly-regulated system and biases the cells toward poorly-directed movements, perhaps because of dysregulation of the timing or function of the mechanisms that control the stability of AJ proteins. This failure in precise regulation of adhesion was also highlighted in the inability of cone cells to exchange their membrane contacts: the apical interfaces of pyd-RNAi expressing cone cells were locked in place. Ectopic DE-Cadherin further increased the percentage of ommatidia affected, again emphasizing the link between pyd activity and the AJ (Seppa, 2008).
The localization of Pyd to the AJ in the pupal eye is dependent on both DE-Cadherin and α-Catenin. However, it was found that ectopic expression of either junctional protein is not sufficient to alter the localization of Pyd. Taken together, these data indicate that DE-Cadherin and α-Catenin are necessary to build or maintain the AJ and to localize Pyd but that, in excess, they are not sufficient to attract ectopic Pyd. This suggests that either Pyd protein levels are not easily altered or that Pyd may be binding to proteins other than the core AJ constituents. Recent work demonstrated that E-Cadherin was necessary for the initial steps of AJ formation while α-Catenin was essential for both the establishment and maintenance of the junction; only when α-Catenin was reduced was ZO-1 lost from established junctions. The results suggest that in dynamically restructured tissues such as the eye, both E-Cadherin and α-Catenin are necessary for the localization of AJ-associated proteins (Seppa, 2008).
The immunoglobulin superfamily member Roughest is necessary for appropriate sorting of IPCs during pupal eye development. Reducing Pyd increased Roughest protein levels specifically at the AJ. Roughest is the Drosophila orthologue of Neph1, a cell adhesion molecule necessary for the structure and function of the glomerular slit diaphragm in the mammalian kidney. The slit diaphragm is the main size-selective barrier in the filtration apparatus of the kidney and retains many characteristics of both the tight and AJ complexes from which it was derived. The Hibris orthologue Nephrin also forms part of the physical structure of the slit diaphragm and both cell adhesion molecules have been reported to bind to each other as well as to ZO-1. Perhaps ZO-1, as with Pyd, has a role in regulating the localization or levels of cell adhesion molecules such as Neph1 and Nephrin (Seppa, 2008).
The Dpp pathway has emerged as a major contributor to patterning of the Drosophila pupal eye. Its role requires functional connections to both DE-Cadherin and Roughest. For example, mutations in shotgun (the locus that encodes DE-Cadherin) suppresses the roughest eye phenotype but enhances Dpp pathway-dependent phenotypes in the eye and wing. Together, these data suggest a model in which (1) Roughest acts to promote the stability of membrane contacts to drive directed cell movements and (2) the Dpp pathway and Pyd act to destabilize the adherens junction complex and local cell contacts to allow for proper IPC sorting. Consistent with this view, it was observed that reducing pyd enhances the effects of reduced Dpp pathway activity in the eye and wing. Thus, Pyd appears to act in concert with the Dpp pathway to regulate select core components of the AJ during development (Seppa, 2008).
This study has shown that Pyd is required specifically for patterning the interommatidial cells of the Drosophila pupal eye. Pyd appears to regulate both cell shape and cell positioning by controlling the levels of AJ proteins such as DE-Cadherin and adhesion proteins such as Roughest. Thus, Pyd provides a link between adhesion and junction formation; a further understanding of its role in the pupal eye will shed light on how these processes are coordinated to generate precise cellular movements during epithelial patterning (Seppa, 2008).
Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained as AJ are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through DE-cadherin endocytosis by limiting the Cdc42/Par6/aPKC complex activity. This study demonstrates that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin containing Rab11-positive recycling endosomes in Drosophila post-mitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 may regulate the formation of recycling endosomes thorough localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin containing vesicular trafficking during AJ remodeling in live epithelia (Yashiro, 2014).
Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, the contraction of actomyosin cables formed in apoptotic and neighboring cells drive cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. A model is proposed in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion, and would further influence biochemical signals of neighboring non-apoptotic cells (Teng, 2016).
This study reports the temporal sequence of events during apoptotic cell extrusion, with a focus on the remodeling of AJs, the cytoskeleton, and mechanical tension. After caspase-3 starts to be activated in the polyploid larval epithelial cells (LECs), those undergoing apoptosis initiate apical constriction. It was reasoned that the initiation of this constriction could be due to a combination of actomyosin cable formation in the dying cell and the activity of caspase-3, which assists in the upregulation of actomyosin contractility. Indeed, it has been shown in tissue culture that the cleavage of Rho associated kinase by caspase- 3 is involved in phosphorylation and activation of myosin light chain, which regulates actomyosin contractility. It is proposed that the actomyosin cable that forms in apoptotic LECs is responsible for the early stages of apoptotic cell extrusion. During apical constriction, the level of AJ components including E-cad strongly reduced in a caspase-3-dependent manner. In the neighboring non- dying cells, this reduction is found only at the interface between the apoptotic cell and its neighbors. Since caspase-3 is not activated in the neighboring cells, it is speculated that the reduction of E-cad is a consequence of a loss of trans-interactions between E-cad of the neighboring cell, and E-cad of the apoptotic cell, which undergoes caspase-3-dependent cleavage. This often, but not always, leads to plasma membrane separation, which is suggestive of a loosening of AJ-dependent adhesion. It has been reported that anillin organizes and stabilizes actomyosin contractile rings at AJs and its knock-down is associated with a reduction of E-cad and β-Catenin levels at AJs, leading to AJ disengagement. A gradual decrease in the level of E-cad, and a gradual increase in MyoII accumulation
in apoptotic cells was observed prior to the strong reduction of E-cad levels. This lead to the hypothesis that mechanical tension exerted on the cell interface between apoptotic LECs and neighboring cells by the contraction of the actomyosin cable, which forms in the apoptotic cell, is large enough to rupture the weakened contacts between plasma membranes at AJs upon the strong reduction of E-cad levels (Teng, 2016).
Interestingly, and by contrast, there are cases when AJs are not disengaged even after the level of E-cad is reduced. In these cases the cells exhibit a separation of actomyosin cables from the membrane. It is speculated that the state of cell-cell contacts at AJs, i.e., whether they will disengage or remain engaged during apoptosis, is dependent on which of the following links is weaker: The link between two plasma membranes, or the link between the plasma membrane and the actomyosin cable. Both of these links would be weakened by a strong, albeit incomplete, reduction of E-cad levels. When the former is weaker than the latter, the two plasma membranes could be detached. When the former is stronger than the latter, the two plasma membranes could remain in contact, and the actomyosin cable could be detached from the plasma membrane (Teng, 2016).
In parallel with the reduction of E-cad levels and the associated release of tension, a supra-cellular actomyosin cable begins to form in neighboring cells. These observations prompted a speculation that the release of tissue tension triggers MyoII accumulation in neighboring cells. Subsequent contraction of this outer ring helps to reshape tissue tension, which is transiently released when E-cad is reduced. As a consequence, the neighboring cells are stretched. Upon
completion of apical constriction, neighboring non-apoptotic cells form de novo AJs and the stretched cells undergo cell division and/or cell-cell contact rearrangement. These processes allow a relaxation of the high tension associated with the stretching of cells. Finally, measurements of caspase-3 activity, and the observations from caspase inhibition experiments, lead to a conclusion that the characteristics associated with apoptotic cell extrusion reported in this study are the consequences of the apoptotic process, rather than the cause (Teng, 2016).
In addition to the progressive remodeling of AJs and modulation of tissue tension during apoptosis, the mechanical role was examined of apoptosis 'apoptotic force' in tissue morphogenesis, which has been proposed, demonstrated, and discussed. It was shown that the mechanical force generated by the contraction of actomyosin cables formed when LECs undergo apoptosis, especially boundary LECs, promotes tissue expansion, along with histoblast proliferation and migration. Nonetheless, it cannot be ruled out that this apical contraction is in part driven by a decrease
in cell volume, which can be triggered by caspase activation. Intriguingly, it was found that apoptosis of non-boundary LECs did not affect tissue
expansion. This raised the possibility that the mechanical influence of apoptosis in neighboring tissues is dependent not only on the physical connections between cells, but also on the mechanical properties of cells, including cell compliance. If a tissue is soft, for instance, the tensile forces generated by apoptotic process could be absorbed by nearest-neighbor cells and would not propagate to cells further than a single cell away. It is speculated that the apoptotic process could mechanically contribute to cell death-related morphogenesis, only when apoptosis takes place at optimal mechanical properties of a tissue (Teng, 2016).
This study presents a framework for understanding how cell adhesions and tissue tension are progressively modulated during apoptosis in a developing epithelium. It is concluded that tissue tension reshaping, including the transient release of tension upon a reduction in the levels of AJ components, represents a mechanism of apoptotic cell extrusion. It would be important to explore how this transient modulation in mechanical tension would further influence the biochemical nature of neighboring non-apoptotic cells (Teng, 2016).
Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from
their precursors are defined. In particular, distinct zones of hemocyte maturation,
signaling and proliferation in the lymph gland during hematopoietic progression
are described. Different stages of hemocyte development have been classified
according to marker expression and placed within developmental niches: a
medullary zone for quiescent prohemocytes, a cortical zone for maturing
hemocytes and a zone called the posterior signaling center for specialized
signaling hemocytes. This establishes a framework for the identification of
Drosophila blood cells, at various stages of maturation, and provides a genetic
basis for spatial and temporal events that govern hemocyte development. The
cellular events identified in this analysis further establish Drosophila as a
model system for hematopoiesis (Jung, 2005).
In the late embryo, the lymph gland consists of a single pair of lobes
containing ~20 cells each. These express the transcription factors Srp and
Odd skipped (Odd),
and each cluster of hemocyte precursors is followed by a string of
Odd-expressing pericardial cells that are proposed to have nephrocyte
function. These lymph gland lobes are arranged bilaterally such that they
flank the dorsal vessel, the simple aorta/heart tube of the open circulatory
system, at the midline. By the second larval instar, lymph gland morphology is
distinctly different in that two or three new pairs of posterior lobes have
formed and the primary lobes have increased in size approximately tenfold (to
~200 cells. By the late third instar, the lymph gland has grown significantly in size
(approximately another tenfold) but the arrangement of the lobes and
pericardial cells has remained the same. The cells of the third instar lymph
gland continue to express Srp (Jung, 2005).
The third instar lymph gland also exhibits a strong, branching network of
extracellular matrix (ECM) throughout the primary lobe. This network was
visualized using several GFP-trap lines in which GFP is fused to endogenous
proteins. For
example, line G454 represents an insertion into the viking
locus, which encodes a Collagen IV component of the extracellular matrix.
The hemocytes in the primary lobes of G454
(expressing Viking-GFP) appear to be clustered into small populations within
pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as
the uncharacterized GFP-trap line ZCL2867, also highlight this
branching pattern. What role this intricate ECM network plays in
hematopoiesis, as well as why multiple cells cluster within these ECM
chambers, remains to be determined (Jung, 2005).
Careful examination of dissected, late third-instar lymph glands by
differential interference contrast (DIC) microscopy revealed the presence of
two structurally distinct regions within the primary lymph gland lobes that
have not been previously described. The periphery of the primary lobe generally exhibits a
granular appearance, whereas the medial region looks smooth and compact. These
characteristics were examined further with confocal microscopy using a
GFP-trap line G147, in which GFP is fused to a microtubule-associated
protein. The G147 line is expressed throughout the lymph gland but, in
contrast to nuclear markers such as Srp and Odd, distinguishes morphological
differences among cells because the GFP-fusion protein is expressed in the
cytoplasm in association with the microtubule network. Cells in the
periphery of the lymph gland make relatively few cell-cell contacts, thereby
giving rise to gaps and voids among the cells within this region. This
cellular individualization is consistent with the granularity of the
peripheral region observed by DIC microscopy. By contrast, cells
in the medial region were relatively compact with minimal intercellular space,
which is also consistent with the smoother appearance of this region by DIC
microscopy. Thus, in the late third instar, the lymph gland primary lobes
consist of two physically distinct regions: a medial region consisting of
compactly arranged cells, which was termed the medullary zone; and a peripheral
region of loosely arranged cells, termed the cortical zone (Jung, 2005).
Mature hemocytes have been shown to express several markers, including
collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter
Collagen-gal4 (Cg-gal4), which is
expressed by both plasmatocytes and crystal cells, is restricted to the
periphery of the primary lymph gland lobe. Comparison of
Cg-gal4 expression in G147 lymph glands, in which the
medullary zone and cortical zone can be distinguished, reveals that maturing
hemocytes are restricted to the cortical zone. In fact,
the expression of each of the maturation markers mentioned above is found to
be restricted to the cortical zone. The reporter hml-gal4 and Pxn,
which are expressed by the plasmatocyte and crystal cell lineages, are
extensively expressed in this region. Likewise,
the expression of the crystal cell lineage marker
Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the
cortical zone was verified by several means, including the distribution of
melanized lymph gland crystal cells in the Black cells background and analysis of the
terminal marker ProPOA1. The cortical zone is also the site of P1
antigen expression, a
marker of the plasmatocyte lineage. The uncharacterized GFP fusion line
ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that
the homeobox transcription factor Cut is
preferentially expressed in the cortical zone of the primary lobe. Although the role of
Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut
are known to be regulators of the myeloid hematopoietic lineage in both mice
and humans. Cells of the rare third cell type, lamellocytes, are also
restricted to the cortical zone, based upon cell morphology and the expression of a
msn-lacZ reporter (msn06946). In summary, based
on the expression patterns of several genetic markers that identify the three
major blood cell lineages, it is proposed that the cortical zone is a specific
site for hemocyte maturation (Jung, 2005).
The medullary zone was initially defined by structural characteristics and subsequently by
the lack of expression of mature hemocyte markers. However, several markers have been
identified that are exclusively expressed in the medullary
zone at high levels but not the cortical zone. Consistent with the compact
arrangement of cells in the medullary zone, it was found that Drosophila
E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant
expression of DE-cadherin was observed among maturing cells in the cortical
zone. E-cadherin, in both vertebrates and Drosophila, is a
Ca2+-dependent, homotypic adhesion molecule often expressed by
epithelial cells and is a crucial component of adherens junctions.
Attempts to study DE-cadherin mutant clones in the medullary zone
where the protein is expressed were unsuccessful since no clones were
recoverable. The reporter lines domeless-gal4 and
unpaired3-gal4 are preferentially expressed in the medullary zone. The gene
domeless (dome) encodes a receptor molecule known to mediate
the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The
unpaired3 (upd3) gene encodes a protein with homology to
Unpaired and has been associated with innate immune function. These
gal4 lines are in this study only as markers that correlate with the
medullary zone and, at the present time, there is no evidence that their
associated proteins have a role in lymph gland hematopoiesis. Other markers of
interest with preferential expression in the medullary zone include the
molecularly uncharacterized GFP-trap line ZCL2897 and
actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary
zone. It is therefore reasonable to propose that this zone is largely
populated by prohemocytes that will later mature in the cortical zone.
Prohemocytes are characterized by their lack of maturation markers, as well as
their expression of several markers described as expressed in the medullary zone (Jung, 2005).
The posterior signaling center (PSC), a small cluster of cells at the posterior
tip of each of the primary (anterior-most) lymph gland lobes,
is defined by its expression of the Notch ligand Serrate and
the transcription factor Collier.
During this analysis, several additional markers were identified that exhibit
specific or preferential expression in the PSC region. For example, it was found
that the reporter Dorothy-gal4 is
strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which
belongs to a class of enzymes that function in the detoxification of
metabolites. The upd3-gal4 reporter, which has preferential
expression in the medullary zone, is also strongly expressed among cells of
the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and
ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has
made it clear that the PSC is a distinct zone of cells that can be defined by
the expression of multiple gene products (Jung, 2005).
The PSC can be defined just as definitively by the
characteristic absence of several markers. For example, the RTK receptor Pvr,
which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise,
dome-gal4 is not expressed in the PSC, further suggesting
that this population of cells is biased toward the production of ligands
rather than receptor proteins. Maturation markers such as Cg-gal4,
which are expressed throughout the cortical zone, are not
expressed by PSC cells. Additionally, the expression levels of the
hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are
dramatically reduced in the PSC when compared with other hemocytes of the
lymph gland. Taken
together, both the expression and lack of expression of a number of genetic
markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).
In contrast to primary lobes of the third instar, maturing hemocytes are
generally not seen in the secondary lobes. Correspondingly, secondary lobes
often have a smooth and compact appearance, much like the
medullary zone of the primary lobe. Consistent with this appearance, secondary
lymph gland lobes also express high levels of DE-cadherin. The size of the
secondary lobe, however, varies from animal to animal and this correlates with
the presence or absence of maturation markers. Smaller secondary lobes contain
a few or no cells expressing maturation markers, whereas larger secondary
lobes usually exhibit groups of differentiating cells. Direct comparison of
DE-cadherin expression in secondary lobes with that of Cg-gal4,
hml-gal4 or Lz revealed that the expression of these maturation markers
occurs only in areas in which DE-cadherin is downregulated. Therefore,
although there is no apparent distinction between cortical and medullary zones
in differentiating secondary lobes, there is a significant correlation between
the expression of maturation markers and the downregulation of DE-cadherin, as
is observed in primary lobes (Jung, 2005).
The relatively late 'snapshot' of lymph gland development in the third
larval instar establishes the existence of spatial zones within the lymph
gland that are characterized by differences in structure as well as gene
expression. In order
to understand how these zones form over time, lymph glands of second instar
larvae, the earliest time at which it was possible to dissect and stain, were
examined for the expression of hematopoietic markers. As expected, Srp and Odd
are expressed throughout the lymph gland during the second instar since they are in the
late embryo and third instar lymph gland. Likewise, the
hemocyte-specific marker Hemese is expressed throughout the lymph gland at
this stage, although it is not present in the embryonic lymph gland (Jung, 2005).
To determine whether the cortical zone is already formed or forming in
second instar lymph glands, the expression of various maturation
markers were examined in a pair-wise manner to establish their temporal order. Of the
markers examined, hml-gal4 and Pxn are the earliest to
be expressed. The majority of maturing cells were found to be double-positive
for hml-gal4 and Pxn expression, although a few cells were found to
express either hml-gal4 or Pxn alone. This indicates that
the expression of these markers is initiated at approximately the same time,
although probably independently, during lymph gland development. The marker
Cg-gal4 is next to be expressed since it was found among a subpopulation
of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in
the early third instar. Interestingly, the early expression of each of these
maturation markers is restricted to the periphery of the primary lymph gland
lobe, indicating that the cortical zone begins to form in this position in the
second instar. Whenever possible, each genetic marker was directly compared
with other pertinent markers in double-labeling experiments, except in cases
such as the comparison of two different gal4 reporter lines or when
available antibodies were generated in the same animal. In such cases, the
relationship between the two markers, for example dome-gal4 and
hml-gal4, was inferred from independent comparison with a third
marker such as Pxn (Jung, 2005).
By studying the temporal sequence of expression of hemocyte-specific
markers, one can describe stages in the maturation of a hemocyte. It should be noted,
however, that not all hemocytes of a particular lineage are identical. For
example, in the late third instar lymph gland, the large majority of mature
plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the
remainder express only Pxn (~15%) or hml-gal4 (~5%) alone.
Thus, while plasmatocytes as a group can be characterized by the expression of
representative markers, populations expressing subsets of these markers indeed exist.
It remains unclear at this time whether this heterogeneity in the hemocyte
population is reflective of specific functional differences (Jung, 2005).
In the third instar, Pxn is a prototypical hemocyte maturation marker,
while immature cells of the medullary zone express dome-gal4.
Comparing the expression of these two markers in the second instar reveals an
interesting developmental progression. A group of cells
along the peripheral edge of these early lymph glands already express Pxn.
These developing hemocytes downregulate the expression of dome-gal4, as they do
in the third instar. Next to these developing hemocytes is a group of cells
that expresses dome-gal4 but not Pxn; these cells are most similar to
medullary zone cells of the third instar and are therefore prohemocytes.
Interestingly, there also exists a group of cells in the second instar that
expresses neither Pxn nor dome-gal4. This population is most easily
seen in the medial parts of the gland, close to the centrally placed dorsal. These
cells resemble earlier precursors in the embryo, except they express the
marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data
is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes.
As prohemocytes begin to mature into hemocytes, dome-gal4 expression
is downregulated, while the expression of maturation markers is initiated. The
prohemocyte and hemocyte populations continue to be represented in the third
instar as components of the medullary and cortical zones, respectively (Jung, 2005).
The cells of the PSC are already distinguishable in the late embryo by
their expression of collier. It was found that the canonical
PSC marker Ser-lacZ is not expressed in the
embryonic lymph gland and is only expressed in a small number of
cells in the second instar. This relatively late onset of expression is consistent with
collier acting genetically upstream of Ser.
Another finding was that the earliest expression of upd3-gal4
parallels the expression of Ser-lacZ and is restricted to the PSC
region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar,
similar to what is seen in the third instar (Jung, 2005).
To determine whether maturing cortical zone cells are indeed derived from
medullary zone prohemocytes, a lineage-tracing experiment was performed in
which dome-gal4 was used to initiate the permanent marking of all
daughter cell lineages. In this system, the dome-gal4 reporter expresses
both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening
FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ
under the control of the actin5C promoter. At
any developmental time point, GFP is expressed in cells where
dome-gal4 is active, while lacZ is expressed in all
subsequent daughter cells regardless of whether they continue to express
dome-gal4. In this experiment, cortical zone cells
are permanently marked with ß-galactosidase despite not expressing
dome-gal4 (as assessed by GFP), indicating that these cells are
derived from a dome-gal4-positive precursor. This result is
consistent with and further supports independent marker analysis that
shows that dome-gal4-positive prohemocytes downregulate
dome-gal4 expression as they initiate expression of maturation
markers representative of cortical zone cells. As controls to the above
experiment, the expression patterns of two other gal4
lines, twist-gal4 and Serrate-gal4 were determined. The reporter
twist-gal4 is expressed throughout the embryonic mesoderm from which
the lymph gland is derived. Accordingly, the entire lymph gland is permanently
marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the
third instar lymph gland. Analysis of Ser-gal4 reveals that PSC
cells remain a distinct population of signaling cells that do not contribute
to the cortical zone (Jung, 2005).
Genetic manipulation of Pvr function provides valuable insight into its
involvement in the regulation of temporal events of lymph gland development.
To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were
generated in the lymph gland early in the first instar and then examined
during the third instar for the expression of maturation markers. It was found
that loss of Pvr function abolishes P1 antigen and Pxn expression,
but not Hemese expression. The crystal cell markers Lz and ProPOA1
are also expressed normally in Pvr-mutant clones,
consistent with the observation that mature crystal cells lack or downregulate
Pvr. The fact that Pvr-mutant cells express Hemese and
can differentiate into crystal cells suggests that Pvr specifically controls
plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL
positive but do express the hemocyte marker Hemese and can differentiate into crystal
cells, all suggesting that the observed block in plasmatocyte differentiation
within the mutant clone is not due to cell death. Additionally, Pvr-mutant
clones were large and
not significantly different in size from their wild-type twin spots.
Thus, the primary role of Pvr is not in the control of cell
proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same
phenotypic features, confirming that Pvr controls the transition
of Hemese-positive cells to plasmatocyte fate (Jung, 2005).
Entry into S phase was monitored using BrdU incorporation and
distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In
the second instar, proliferating cells are evenly distributed throughout the
lymph gland. By the
third instar, however, the distribution of proliferating cells is no longer
uniform; S-phase cells are largely restricted to the cortical zone. This is
particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary
zone cells, which can be identified by the expression of dome-gal4,
rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second
instar lymph gland quiesce as they populate the medullary zone of the third
instar. As prohemocytes transition into hemocyte fates in the cortical zone,
they once again begin to expand in number. This is supported by the
observation that the medullary zone in white pre-pupae does not appear
diminished in size, suggesting that the primary mechanism for the
expansion of the cortical zone prior to this stage is through cell division
within the zone. Proliferating cells in the secondary lobes continue to be
distributed uniformly in the third instar, suggesting that
secondary-lobe prohemocytes do not reach a state of quiescence as do the cells
of the medullary zone. These results indicate that cells of the lymph gland go
through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).
This analysis of the lymph gland revealed three key features that arise
during development. The first feature is the presence of three distinct zones
in the primary lymph gland lobe of third instar larvae. Two of these zones,
termed the cortical and medullary zones, exhibit structural
characteristics that make them morphologically distinct. These zones, as well
as the third zone, the PSC, are also distinguishable by the expression of
specific markers. The second key feature is that cells expressing
maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and
Cg-gal4 are restricted to the cortical zone. The medullary zone is
consistently devoid of maturation marker expression and is therefore defined
as a region composed of immature hemocytes (prohemocytes). The finding of
different developmental populations within the lymph gland (prohemoctyes and
their derived hemocytes) is similar to the situation in vertebrates where it
is known that hematopoietic stem cells and other blood precursors give rise to
various mature cell types. Additionally, Drosophila hemocyte
maturation is akin to the progressive maturation of myeloid and lymphoid
lineages in vertebrate hematopoiesis. The third key feature of lymph gland
hematopoiesis is the dynamic pattern of cellular proliferation observed in the
third instar. At this stage, the vast majority of S-phase cells in the primary
lobe are located in the cortical zone, suggesting a strong correlation between
proliferation and hemocyte differentiation. Compared with earlier
developmental stages, cell proliferation in the medullary zone actually
decreases by the late third instar, suggesting that these cells have entered a
quiescent state. Thus, proliferation in the lymph gland appears to be
regulated such that growth, quiescence and expansion phases are evident
throughout its development (Jung, 2005).
Drosophila blood cell precursors, prohemocytes and maturing
hemocytes each exhibit extensive phases of proliferation. The competence of
these cells to proliferate seems to be a distinct cellular characteristic that
is superimposed upon the intrinsic maturation program. Based on the patterns
of BrdU incorporation in developing primary and secondary lymph gland lobes,
it is possible to envision at least two levels of proliferation control during
hematopoiesis. It is proposed that the widespread cell proliferation observed in
second instar lymph glands and in secondary lobes of third instar lymph glands
occurs in response to a growth requirement that provides a sufficient number
of prohemocytes for subsequent differentiation. The mechanisms promoting
differentiation in the cortical zone also trigger cell proliferation, which
accounts for the observed BrdU incorporation in this zone and serves to expand
the effector hemocyte population. The quiescent cells of the medullary zone
represent a pluripotent precursor population because they, similar to
vertebrate hematopoietic precursors, rarely divide and give rise to multiple
lineages and cell types (Jung, 2005).
Based on this analysis a model is proposed by which
hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland
are first distinguishable as Srp+, Odd+ (S+O+) cells. These will
eventually give rise to a primary lymph gland lobe where the steps of hemocyte
maturation are most apparent. During the first or early second instar, these
S+O+ cells begin to express the hemocyte-specific marker
Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called
pre-prohemocytes and, in the second instar, cells expressing only these
markers occupy a narrow region near the dorsal vessel. Subsequently, a subset
of these Srp+, Odd+, He+, Pvr+
(S+O+H+Pv+) pre-prohemocytes
initiate the expression of dome-gal4 (dg4),
thereby maturing into prohemocytes. The prohemocyte population
(S+O+H+Pv+dg4+)
can be subdivided into two developmental stages. Stage 1 prohemocytes, which
are abundantly seen in the second instar, are proliferative, whereas stage 2
prohemocytes, exemplified by the cells of the medullary zone, are quiescent.
As development continues, prohemocytes begin to downregulate
dome-gal4 and express maturation markers (M; becoming
S+O+H+Pv+dg4lowM+).
Eventually, dome-gal4 expression is lost entirely in these cells
(becoming
S+O+H+Pv+dg4-M+),
found generally in the cortical zone. Thus, the maturing hemocytes of the
cortical zone are derived from prohemocytes previously belonging to the
medullary zone. This is supported by lineage-tracing experiments that show
cells expressing medullary zone markers can indeed give rise to cells of the
cortical zone. In turn, the medullary zone is derived from the earlier,
pre-prohemocytes. Early cortical zone cells continue to express successive
maturation markers (M) as they proceed towards terminal differentiation. Depending on the
hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1,
msn-lacZ, etc. These studies have shown that differentiation of the
plasmatocyte lineage requires Pvr, while previous work has shown that the
Notch pathway is crucial for the crystal cell fate. Both
the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).
Previous investigations have demonstrated that similar transcription
factors and signal transduction pathways are used in the specification of
blood lineages in both vertebrates and Drosophila. Given this
relationship, Drosophila represents a powerful system for identifying
genes crucial to the hematopoietic process that are conserved in the
vertebrate system. The work presented here provides an analysis of
hematopoietic development in the Drosophila lymph gland that not only
identifies stage-specific markers, but also reveals developmental mechanisms
underlying hemocyte specification and maturation. The prohemocyte population
in Drosophila becomes mitotically quiescent, much as their
multipotent precursor counterparts in mammalian systems. These conserved
mechanisms further establish Drosophila as an excellent genetic model
for the study of hematopoiesis (Jung, 2005).
Mechanisms that govern cell-fate specification within developing epithelia have been intensely investigated, with many of the critical intercellular signaling pathways identified, and well characterized. Much less is known, however, about downstream events that drive the morphological differentiation of these cells, once their fate has been determined. In the Drosophila wing-blade epithelium, two cell types predominate: vein and intervein. After cell proliferation is complete and adhesive cell-cell contacts have been refined, the vast majority of intervein cells adopt a hexagonal morphology. Within vein territories, however, cell-shape refinement results in trapezoids. Signaling events that differentiate between vein and intervein cell fates are well understood, but the genetic pathways underlying vein/intervein cyto-architectural differences remain largely undescribed. This study shows that the Rap1 (Roughened) GTPase plays a critical role in determining cell-type-specific morphologies within the developing wing epithelium. Rap1, together with its effector Canoe, promotes symmetric distribution of the adhesion molecule DE-cadherin about the apicolateral circumference of epithelial cells. Evidence is provided that in presumptive vein tissue Rap1/Canoe activity is down-regulated, resulting in adhesive asymmetries and non-hexagonal cell morphologies. In particular Canoe levels are reduced in vein cells as they morphologically differentiate. It was also demonstrate that over-expression of Rap1 disrupts vein formation both in the developing epithelium and the adult wing blade. Therefore, vein/intervein morphological differences result, at least in part, from the patterned regulation of Rap1 activity (O'Keefe, 2012).
During the early, proliferative phase of epithelial development each cell strives to maintain adhesive contacts with its neighbors, generating, on average, a field of hexagonal-shaped cells. This uniformity is transient, however, as multiple cell types are frequently specified within a single epithelium, each with a unique function and cyto-architecture. Mechanisms must exist, therefore, for cell-type-specific shapes to emerge as these heterogeneous epithelia begin to morphologically differentiate. This study shows that in the Drosophila wing the regulation of Rap1 activity is one means by which non-hexagonal epithelial cell shapes are generated (O'Keefe, 2012).
These studies have focused on the Drosophila wing vein. Within the wing blade, veins comprise a small subset of cells, and during pupal stages of development it was shown that vein-precursor cells adopt a unique shape (trapezoidal), compared to surrounding intervein cells (hexagonal). Presumptive vein cells are first identified by high levels of Egfr activity, and previous studies have shown that Egfr signaling up-regulates the homophilic adhesion molecule DE-cad in these cells (both transcriptionally and post-translationally) (O'Keefe, 2007). High levels of cadherin generally result in apical constriction, a prominent characteristic of the adult vein. DE-cad is only one component of this morphogenetic process, however, as increased levels of DE-cad did not result in a vein-like trapezoidal shape. It was asked, therefore, what other mechanisms might determine the non-hexagonal morphology of vein precursors (O'Keefe, 2012).
In addition to elevated levels of DE-cad, another distinguishing feature of pupal vein cells is an asymmetric distribution of DE-cad about their apicolateral circumference, a phenotype most apparent when two-cell clones of ectopic veins were examined. As loss of Rap1 leads to asymmetric DE-cad (Knox, 2002; O'Keefe, 2009), it was hypothesized that Rap1 activity is down-regulated in vein precursor cells compared to surrounding intervein precursors. Consistent with this hypothesis, Rap1 over-expression dramatically disrupted pupal vein cell shape without affecting cell fate (i.e., DSRF levels). Rap1 over-expressing vein cells had more symmetric DE-Cad distributions, and did not adopt a trapezoidal morphology. This often led to morphological vein defects in the adult wing. In addition, the localization patterns of Rap1-GFP and Canoe suggested lower levels of Rap1 activity in pupal-vein precursors (compared with surrounding intervein cells). It has been previously demonstrated that the generation of Rap1 loss-of-function clones during larval stages results in vein loss (O'Keefe, 2009). Rap1 activity, therefore, plays a dual role in wing-vein formation. First, during larval and early pupal stages, Rap1 stabilizes adhesive contacts between adjacent epithelial cells, thereby facilitating Egfr signaling and maintaining vein-cell fate. Hours later, as the wing begins to differentiate, down-regulation of Rap1 activity drives the morphological changes necessary for vein formation (O'Keefe, 2012).
How does the down-regulation of Rap1 activity specifically increase DE-cad levels at vein-vein cell contacts? Rap1 recruits Cno to adherens junctions, where Cno forms a physical link between adherens junctions and the actin cytoskeleton (Sawyer, 2009). As such, Cno primarily acts as a non-enzymatic scaffolding protein, which suggests that stoichiometry between DE-cad and Cno is important. Based on immunofluorescence analysis of apicolateral cell junctions in the wing, there is a large disparity between Cno and DE-cad levels in vein cells, as Egfr/Ras signaling both up-regulates DE-cad, and down-regulates Cno. It is inferred from these data that vein cells contain far fewer adherens junction complexes that are associated with a molecule(s) of Cno (compared to intervein cells). As Cno represents the critical Rap1 effector in this context, these Cno-free adherens junction complexes would be functionally dissociated from Rap1 signaling, and free to localize in an asymmetric fashion. Relieved from spatial constraints concerning symmetry, adherens junction complexes would accumulate at vein-vein interfaces, where chances of encountering an intercellular binding partner are highest for two reasons: 1) adjacent vein cells express higher levels of DE-cad than adjacent intervein cells, and 2) adjacent vein cells contain Cno-free adherens junction complexes, which are similarly relieved from symmetry constraints (O'Keefe, 2012).
The formation of asymmetrical adhesive contacts in presumptive vein cells is coincident with changes in apical cell shape. It was asked, therefore, how changes in DE-cad localization might affect vein-cell shape, and have proposed a simple model based on examinations of a timecourse of vein differentiation. The balance between intercellular adhesion and cortical tension is a critical determinant of cell shape. Increased adhesion expands cell contacts, and cortical tension opposes this effect. The data suggest that after ~24 h APF, vein-vein cell contacts are characterized by high levels of adhesion (i.e., DE-cad) and decreased levels of cortical tension (i.e., Cno, which links adherens junctions to the actin cytoskeleton). It is hypothesized that these factors drive the expansion of vein-vein contacts at the expense of one vein-intervein cell contact, resulting in the formation of a pentagon. Real-time imaging of vein differentiation will be used in the future to test this model of morphogenesis (O'Keefe, 2012).
The Egfr/Ras and Dpp signaling pathways act in concert to specify vein-cell fate. At 12-16 h APF, Egfr/Ras activity turns on dpp expression in presumptive vein cells. After this stage of development, Dpp is required to maintain vein identity and high levels of Egfr/Ras signaling in presumptive vein cells (creating a positive feed-back loop). In contrast, these developmental signaling pathways have very different effects on cell adhesion and epithelial cell morphology. It has been shown previously that Egfr/Ras activity up-regulates DE-cad levels in vein precursors, and that it does so in a Dpp-independent fashion (O'Keefe, 2007). Results presented in this study indicate that Egfr/Ras signaling also plays the dominant role in regulating Rap1/Cno. Two-cell clones that express RasV12 phenotypically resembled Rap1 loss-of-function cells (more so than TkvQ235D clones). In addition, RasV12 down-regulated the critical Rap1 effector Cno, whereas this effect was not evident in TkvQ235D-expressing cells. As loss of Cno disassociates actin-myosin contractility from cell shape (Sawyer, 2009), RasV12 two-cell clones were less apically constricted than TkvQ235D-expressing cells. Egfr/Ras signaling is also associated with asymmetric adhesive contacts in other developmental contexts. In the Drosophila eye, for example, Egfr/Ras signaling is required in photoreceptors. Much like vein cells, photoreceptors adhere more tightly to one another than to surrounding cells. This raises the possibility that Egfr down-regulates Rap1 activity in multiple cell types following their specification, enabling them to differentiate appropriate cell shapes. Finally, it will be interesting to determine how the Egfr/Ras and Dpp signaling pathways regulate other aspects of vein-cell morphology (e.g., constriction along the apical/basal axis to generate a lumen) (O'Keefe, 2012).
In the wing, Egfr/Ras signaling does not affect Rap1/Cno activity at every developmental stage. High levels of Egfr/Ras signaling are detected in vein cells at the beginning of the third larval instar, but vein/intervein cell-shape differences are not observed before ~24 h APF. As such, the Rap1/Cno complex likely represents a pupal-specific target of Egfr signaling. This study has shown, therefore, that a single developmental signaling pathway can first determine a cell's fate, and later contribute towards its morphological differentiation. Critical to this process, therefore, are genetic and/or epigenetic factors that temporally regulate the output of Egfr/Ras signaling. In the future it will be important to identify such factors not only for the Egfr/Ras pathway, but other developmental signaling pathways as well (O'Keefe, 2012).
Finally, it is becoming increasingly clear that Rap1 affects cancer progression, often by promoting metastasis. In cancer cells, levels of Rap1 activity are typically high, which stimulates migration and metastasis by up-regulating integrin-based cell adhesion. Such is the case in pancreatic, prostate, and breast cancers. However, loss of Rap1 can also cause metastasis by down-regulating cadherin and disrupting the epithelial integrity of the tumor (e.g., ovarian and prostate cancer). Within this disease context, the Egfr/Ras and Rap1 signaling networks often interact. Most recently, Egfr activation of Rap1 has been shown to promote metastasis of human pancreatic carcinoma cells. The precise mechanisms by which Egfr/Ras signaling affects Rap1 activity (both during normal development and disease) must be deciphered, therefore, if these metastatic processes are to be understood and/or mitigated (O'Keefe, 2012).
In a Drosophila follicle the oocyte always occupies a posterior position among a group of sixteen germline cells. Although the importance of this cell
arrangement for the subsequent formation of the anterior-posterior axis of the embryo is well documented, the molecular mechanism responsible for the
posterior localization of the oocyte has been unknown. The homophilic adhesion molecule Shotgun has now been shown to mediate oocyte positioning. During
follicle biogenesis, Shotgun is expressed in germline (including oocyte) and surrounding follicle cells, with the highest concentration of Shotgun being
found at the interface between oocyte and posterior follicle cells. Mosaic analysis shows that Shotgun is required in both germline and follicle cells for
correct oocyte localization, indicating that germline-soma interactions may be involved in this process. By analysing the behaviour of the oocyte in follicles
with a chimaeric follicular epithelium, the position of the oocyte is seen to be determined by the position of Shotgun-expressing follicle cells, to which the
oocyte attaches itself selectively. Among the Shotgun positive follicle cells, the oocyte preferentially contacts those cells that express higher levels of
Shotgun. On the basis of these data, it is proposed that in wild-type follicles the oocyte competes successfully with its sister germline cells for contact to the
posterior follicle cells, a sorting process driven by different concentrations of Shotgun. This is the first in vivo example of a cell-sorting
process that depends on differential adhesion mediated by a cadherin (Godt, 1998).
The Drosophila gene taiman encodes a steroid hormone receptor coactivator related to AIB1. Mutations in tai cause defects in the migration of specific follicle cells, the border cells, in the Drosophila ovary. Drosophila E-cadherin (Shotgun) is required for border cell migration. To determine whether the tai migration defect might be due to reduction in Shotgun expression, egg chambers containing tai mutant clones were stained with antibodies against Shotgun. In all wild-type stages examined, Shotgun accumulates in the central, nonmigratory polar cells, as well as in the junctions between individual border cells. Shotgun colocalizes with cortical F-actin in these locations. Prior to migration, when the border cells are still part of the follicular epithelium, Shotgun also accumulates at the junctions between border cells and nurse cells. However, once the border cells leave the follicular epithelium and invade the neighboring germline cell cluster, much less Shotgun staining is evident at the junctions between the nurse cells and border cells, relative to the level between border cells or in the polar cells. When migration is complete, Shotgun accumulates again in the junctions between the border cells and the oocyte (Bai, 2000).
In tai mutant clusters, Shotgun staining is abnormally elevated at the border cell/nurse cell junctions. In contrast, in slbo mutants, Shotgun expression fails to rise at the time of migration and Shotgun immunoreactivity is only detected at high levels within the polar cells. Armadillo (Arm) colocalizes with Shotgun in wild-type and mutant border cells. The abnormal accumulation of Shotgun and Arm in tai mutants does not appear to result from increased transcription of Shotgun because overexpression of Shotgun in border cells causes neither a migration defect nor specific accumulation of cadherin staining at the border cell/nurse cell junctions. Nor does the abnormal accumulation of Shotgun and Arm appear to be simply a consequence of the migration failure. In addition to slbo, Shotgun and Arm expression were examined in border cells that fail to migrate due to mutations in the jing locus: no defect in either expression or localization of adhesion complexes was observed. Nor are defects in either Shotgun or Arm expression or localization found in border cells that fail to migrate due to expression of dominant-negative Rac (Bai, 2000).
The accumulation of Shotgun at the border cell/nurse cell boundary suggests that the role of tai in border cell migration might be to stimulate turnover of adhesion complexes during migration in order to allow forward movement. One protein believed to play a role in turnover of adhesion complexes is Focal adhesion kinase. Drosophila FAK (Fak56D) is highly enriched in the border cells during their migration, but not in the polar cells (Bai, 2000).
To determine whether Fak56D expression or localization is affected by mutations that disrupt border cell migration, wild-type and slbo mutant egg chambers were stained and the staining was compared to that of egg chambers containing tai mosaic clones. Fak56D expression is significantly reduced in slbo mutant border cells. Furthermore, the level of reduction correlates with the degree of inhibition of migration. That is, in some slbo egg chambers, border cell migration fails completely and the cells remain at the anterior tip. In such egg chambers, Fak56D expression is undetectable. In a minority of slbo mutant chambers, the cells migrate a little. In these egg chambers, Fak56D expression is reduced compared to wild type, but is detectable. In tai mutant border cells, Fak56D expression is present; however, its distribution is altered relative to wild type. Rather than being evenly distributed throughout the cytoplasm, Fak56D appears to accumulate at the would-be leading edge of the cluster. Some border cell clusters that are mutant for tai exhibit partial migration and in these clusters, the abnormal distribution of Fak56D is only slightly affected such that little Fak56D accumulation can be detected at the most posterior position within the cluster. Thus, the severity of the migration defect in tai mutants correlates with the severity of the defect in Fak56D localization (Bai, 2000).
Follicle cell clones mutant for either Nicastrin (Ncr) or Presenilin (Psn) have a more severe phenotype than that seen in Notch or Delta mutants, indicating that both proteins must have at least one additional function in these cells that is independent of their role in Notch signaling. One aspect of this phenotype is the overaccumulation of the components of the adherens junctions, DE-Cadherin, Armadillo, and alpha-catenin, and this is probably related to the fact that both alpha-catenin and the Armadillo ortholog ß-catenin associate with Psn in mammalian cells. Although neither is required for the activity of the S3 protease or gamma-secretase, loss of Psn leads to an overaccumulation of ß-catenin in Drosophila embryos and mouse epithelial cells. The precise function of Psn in ß-catenin regulation is unknown, but the overexpressed protein in Drosophila psn mutant embryos is associated with polyubiquitin-positive cytoplasmic inclusions, suggesting that Psn is required in some way to regulate Armadillo degradation. Psn also regulates the turnover of DE-Cadherin and alpha-catenin. Furthermore, Nct is necessary for this function, suggesting that it requires the formation of the high molecular weight protease complex. Since Psn is thought to mediate the proteolysis of membrane proteins, one possibility is that Psn is recruited to DE-Cadherin by binding to the catenins, and cleaves DE-Cadherin to trigger degradation. Alternatively, Psn could regulate the turnover of the catenins in some other way, and their overaccumulation in psn and nct mutants might then lead to the stabilization of Cadherin complexes at the membrane (López-Schier, 2002).
Drosophila ovarian follicle stem cells (FSCs) were used to study how stem cells are regulated by external signals. and three main conclusions were drawn. First, the spatial definition of supportive niche positions for FSCs depends on gradients of Hh and JAK-STAT pathway ligands, which emanate from opposite, distant sites. FSC position may be further refined by a preference for low-level Wnt signaling. Second, hyperactivity of supportive signaling pathways can compensate for the absence of the otherwise essential adhesion molecule, DE-cadherin, suggesting a close regulatory connection between niche adhesion and niche signals. Third, FSC behavior is determined largely by summing the inputs of multiple signaling pathways of unequal potencies. Altogether, these findings indicate that a stem cell niche need not be defined by short-range signals and invariant cell contacts; rather, for FSCs, the intersection of gradients of long-range niche signals regulates the longevity, position, number, and competitive behavior of stem cells (Vied, 2012).
Stem cells are generally maintained in appropriate numbers at
defined locations. It is therefore expected that a specific extracellular
environment defines a supportive niche and regulates
stem cell numbers. However, the mechanisms for supporting
and regulating stem cells may vary widely. In the Drosophila germarium, GSCs are principally regulated directly by a single (BMP) pathway that is activated by signals
from immediately adjacent Cap cells and acts within GSCs to
prevent differentiation. This study shows that in the same tissue, FSCs are regulated
by the activity of at least four major signaling pathways, that
the ligands for at least three of these pathways (Wnt, Hh, and
JAK-STAT) derive from distant cells and that these pathways
appear to collaborate in order to define supportive niche positions
for FSCs and the number of FSCs that are supported.
Most crucially, FSCs provide a particularly interesting paradigm
where the intersection of gradients of long-range niche signals
regulates stem cell maintenance, position, number and competitive
behavior (Vied, 2012).
How the strength of a signaling pathway specifies
FSC numbers and supportive niche positions was examined by manipulating
the Hh pathway. Normally, Hh pathway activity is marginally
higher in FSCs than in their daughters and is progressively lower
in more posterior cells, consistent with Hh emanating from Cap
and Terminal Filament cells at the anterior tip of the germarium. Small reductions in
Hh pathway activity led to FSC loss while small increases caused
FSCs to outcompete their neighbors. FSCs must therefore
reside reasonably close to the anterior of the germarium in order
to receive sufficient stimulation by Hh, but what prevents FSCs
from moving progressively further anterior and enjoying even
stronger Hh stimulation? Wg is expressed in anterior Cap cells
along with Hh. Here
it was found that excess Wnt pathway activity strongly impairs
FSC maintenance and that loss of Wnt pathway activity during
FSC establishment can lead to enhanced FSC function and
to a modest accumulation of Wg-insensitive FSC derivatives in
ectopically anterior positions. These observations suggest that
anterior Wg expression contributes to limiting the anterior spread
of FSCs. However, Wg-insensitive cells do not spread to the
extreme anterior of germaria, suggesting that additional factors
control the position of FSCs along the anterior-posterior axis of the germarium (Vied, 2012).
In fact, apparent FSC duplication and anterior movement
of FSC derivatives, including Fas3-negative FSC-like cells,
was seen very dramatically in response to elevated JAK-STAT pathway
activity. Furthermore, the pattern of expression of a reporter of
JAK-STAT pathway activity and its response to localized inhibition
of ligand production showed that the JAK-STAT pathway in
FSCs is activated primarily by ligand emanating from more
posterior, polar cells. Hence, it is suggested that normal FSCs are
unable to function in significantly more anterior positions
because they would receive inadequate stimulation of the JAK-STAT pathway (Vied, 2012).
Thus, the combination of graded distributions of Hh, Wnt, and
JAK-STAT pathway ligands appears to be instrumental in setting
the anterior-posterior position of FSCs and how many FSCs may
be supported in each germarium. Neither the Hh nor the JAK-STAT
pathway activity gradients appear to be classical smooth
gradients but both are high in the central 2a/b region of the
germarium (where FSCs are located) and considerably lower in
either the anterior (JAK-STAT) or posterior (Hh) third of the
germarium. Although FSCs are normally supported by both Hh
and JAK-STAT pathways, JAK-STAT pathway hyperactivity
could substantially compensate for loss of Hh pathway activity
to support FSCs that are neither rapidly lost nor displace wildtype
FSCs. It is therefore concluded that the sum of quantitative
inputs of these two pathways is a key parameter for supporting
normal FSC function (Vied, 2012).
It was first considered that Hh, Wnt, and JAK-STAT pathways
might have a major effect on the migratory or adhesive properties
of FSCs partly because favorable pathway manipulations
led to ectopically positioned FSC-like cells in the germarium
and displacement of wild-type FSCs. It is possible that enhanced
proliferation could also contribute significantly to these phenotypes.
Indeed, FSC proliferation is likely modulated by several
signaling pathways and has been shown to be important for
FSC retention in the niche. However, to date, manipulation of cell proliferation
alone in an FSC has not produced the displacement of
wild-type FSCs that has been observed in response to altered Hh,
JAK-STAT, and Wnt pathways.
Further evidence for FSC signals regulating adhesion comes
from the observation that favorable mutations in all four signaling
pathways that were investigated in this study obviated, to a remarkable degree for Hh
and JAK-STAT pathways, the normal requirement of FSCs for
DE-cadherin function. Again, it is possible that enhanced FSC
proliferation may compensate for defective niche adhesion. In
fact, partial restoration of FSC maintenance has previously
been seen in response to excess Cyclin E or E2F activity for
FSCs lacking a regulator of the actin cytoskeleton likely to
contribute to adhesion. Nevertheless, the
continued retention of FSCs in the germarium despite the
absence of DE-cadherin is most simply explained if Hh and
JAK-STAT pathways alter FSC adhesive properties to favor FSC retention (Vied, 2012).
The cellular interactions guiding the location of FSCs are likely
quite complex, involving prefollicle cells, Escort Cells, germline
cysts and the basement membrane. Some of the observations made in this study
suggest that JAK-STAT signaling might act, in part, by promoting
integrin interactions with the basement membrane. Normally,
laminin A ligand and strong integrin staining along the basement
membrane do not extend further anterior than the FSCs (O'Reilly,
2008). Perhaps excess JAK-STAT signaling facilitates
increasingly anterior deposition of laminin A and organization
of adhesive integrin complexes, promoting simultaneous anterior
migration and basement membrane adhesion of cells of
the FSC lineage. In support of this hypothesis, anterior
extension of integrin staining and apparent anterior migration
of Hop-expressing cells were seen, principally along germarial walls.
However, the requirement or sufficiency of these changes in
integrin organization remains to be tested (Vied, 2012).
For excessive Hh signaling, ectopic cells also often associated
with germarial walls but these cells did not accumulate in far
anterior positions or change the pattern of integrin staining, so
enhanced integrin-mediated associations seem unlikely to
explain the phenotype. The Hh hyperactivity phenotype is very
strong in the absence of DE-cadherin function in FSCs, so
what other adhesive function might be altered by Hh signaling?
Partial restoration of smo mutant FSC maintenance by increased
DE-cadherin expression provides some
further support that adhesive changes are an important component
of the FSC response to Hh. It has been noted that ptc mutant follicle cells rarely contact germline cells in mosaic egg chambers, preferentially occupying positions
between egg chambers or surrounding the follicle cell epithelium, suggesting that excess Hh pathway activity in cells
of the FSC lineage may reduce their affinity for germline cells or
their propensity to integrate into an epithelium. Adhesion to posteriorly
moving germline cysts and a nascent follicular epithelium
would seem a priori to be the major influences tending to pull
FSCs and their daughters away from a stable germarial association.
A reduction in FSC or FSC daughter interactions with
germline cysts or with prefollicle cells might therefore lead to
increased retention of FSCs in the neighborhood of the normal
FSC niche, facilitating accumulation of extra FSCs or allowing
FSC retention even in the absence of DE-cadherin (Vied, 2012).
Most cancers involve signaling pathway mutations and several
such mutations likely originate in stem cells, where selective
pressures may eliminate or amplify mutant cell lineages. It is therefore important to understand how signaling pathways regulate stem cells. The current studies on FSCs
highlight some significant principles that may be widely relevant
to human epithelial cell cancers. First, activating mutations in
signaling pathways normally required for maintenance of the
stem cell in question can amplify the number of stem or stemlike
cells in a local environment. This produces an increased
number of identical but independent genetic lineages, greatly
facilitating the acquisition and selection of secondary mutations
that push a mutant stem cell lineage toward a cancerous phenotype.
Second, signaling pathway mutations can enhance a stem
cell’s ability to compete for niche positions, promoting occupation
of all available niches in an insulated developmental compartment.
These stem cells are now no longer vulnerable to
competition from wild-type stem cells and are effectively immortalized
if, as for FSCs, daughter cells readily replace lost stem
cells. Third, signaling pathway alterations can compensate for
deficits in other pathways or other contributors to normal stem
cell function. Hence, stem cell self-renewal can now tolerate
significant further mutations and changes in their environment
that accompany cancer progression. Loss of epithelial cadherin
function provides a specific example of a significant mutation
that would be expected often to contribute to cancer development
by spurring an epithelial to mesenchymal cell transition,
but which can (in FSCs) only be propagated in stem cells after
mutational hyperactivation of a key signaling pathway. Finally,
these studies emphasize that it is possible for many pathways to
exert strong influences on a single stem cell type; in FSCs, Hh,
JAK-STAT, and PI3K pathway hyperactivity phenotypes are extremely strong, while Wnt and BMP pathways can also play significant roles (Vied, 2012).
Adult stem cells reside in specialized microenvironments, or niches, that have an important role in regulating stem cell behaviour. Therefore, tight control of niche number, size and function is necessary to ensure the proper balance between stem cells and progenitor cells available for tissue homeostasis and wound repair. The stem cell niche in the Drosophila male gonad is located at the tip of the testis where germline and somatic stem cells surround the apical hub, a cluster of approximately 10-15 somatic cells that is required for stem cell self-renewal and maintenance. Somatic stem cells in the Drosophila testis contribute to both the apical hub and the somatic cyst cell lineage. The Drosophila orthologue of epithelial cadherin (DE-cadherin) is required for somatic stem cell maintenance and, consequently, the apical hub. Furthermore, the data indicate that the transcriptional repressor escargot regulates the ability of somatic cells to assume and/or maintain hub cell identity. These data highlight the dynamic relationship between stem cells and the niche and provide insight into genetic programmes that regulate niche size and function to support normal tissue homeostasis and organ regeneration throughout life (Voog, 2008).
Many stem cell niches include support cells that influence stem cell behaviour through secretion of diffusible molecules. Physical contact between stem cells and support cells and/or the extracellular matrix holds stem cells within the niche and close to self-renewal signals. Furthermore, niches provide spatial and mechanical cues that influence the fate of stem cell daughters. Therefore, the stem cell niche has an important role in regulating stem cell maintenance, self-renewal and survival. However, little is known about the factors that regulate niche maintenance or size (Voog, 2008).
Approximately ten somatic cells, called the hub, are found at the apical tip of the Drosophila testis. Germline stem cells (GSCs) and somatic stem cells (SSCs) surround and are in contact with hub cells. Whereas GSCs sustain spermatogenesis, SSCs produce cyst cells that encapsulate the maturing germ cells and ensure differentiation. Hub cells secrete the growth factor Unpaired (Upd), which activates the JAK-STAT signal transduction pathway in adjacent stem cells. JAK-STAT signalling is necessary for stem cell maintenance and is sufficient to specify self-renewal of both GSCs and SSCs in the testis (Voog, 2008).
The apical hub is typically described as a post-mitotic, static structure. However, in agametic flies, SSCs proliferate and express hub markers, leading to an apparent expansion of the apical hub. Furthermore, recent studies have noted that hub cell number and function decrease with age, indicating that the stem cell niche in the testis is dynamic. Hub cells and SSCs share numerous features, including similar gene expression patterns and close association with GSCs and each other; however, the precise relationship between SSCs and hub cells has not been explored (Voog, 2008).
It has been proposed that SSCs may serve as a source of cells that contribute to the apical hub and, consequently, the stem cell niche. To address whether SSCs give rise to hub cells, positively marked beta-galactosidase-expressing (beta-gal+) SSCs were generated using mitotic recombination, a technique typically used for lineage tracing analyses. Labelled SSCs were generated by heat-shocking flies of the appropriate genotype to initiate FLP-mediated recombination, resulting in reconstitution of the alpha-tubulin promoter upstream of the lacZ gene1. Hub cells were identified by immunolabelling with antibodies to Fasciclin III (FasIII)14 or DE-cadherin, cell-surface proteins concentrated at hub cell junctions. SSCs and early cyst cells were identified by immunolabelling with antibodies to Traffic Jam (TJ), a transcription factor that is strongly expressed in early cyst cell nuclei and weakly expressed in hub cells (Voog, 2008).
Wild-type SSC clones were identified as beta-gal+ cells adjacent to the apical hub and surrounding germ cells. A series of heat shocks after eclosion (hatching) led to at least one beta-gal+ SSC in 57% of testes from 3-day-old males analysed 1 day after heat shock. At 5 days after heat shock, 28% of testes contained at least one beta-gal+ SSC, and this frequency decreased to 10%, 10% and 4% at 10, 15 and 30 days after heat shock, respectively (Voog, 2008).
In addition to beta-gal+ SSCs, beta-gal+ hub cells were also observed that co-labelled with DE-cadherin and FasIII. In fact, beta-gal+ cells were found within the hub in 34%, 47% and 60% of testes from males at 1, 5 and 15 days after heat shock, respectively. No beta-gal+ cells were observed in flies not exposed to the heat-shock protocol (Voog, 2008).
Previous reports concluded that hub cells are post-mitotic; however, it is possible that hub cells undergo rare divisions to become marked during recombination. To test whether hub cells are mitotic, dividing cells in the testis were labelled with 5'-bromo-2-deoxyuridine (BrdU), which is incorporated into newly synthesized DNA during S phase. Flies were fed ('pulsed') BrdU for 30 min and aged ('chased') for up to 15 days. Subsequently, labelled testes were co-stained with antibodies to BrdU, as well as to the hub marker FasIII. No BrdU-positive (BrdU+) hub cells were detected after a 1-day chase, although cells adjacent to the hub were clearly labelled (Voog, 2008).
However, BrdU+ hub cells were observed 3-10 days after labelling. BrdU+ hub cells were present in 4%, 8% and 3% of testes assayed at 5, 7-8 and 10 days after labelling. Moreover, these BrdU+ cells co-expressed FasIII and an upd reporter, indicating that these cells function as hub cells. These data are consistent with previous results indicating that hub cells are post-mitotic and support the hypothesis that mitotically active SSCs act as a source of cells that can contribute to the apical hub (Voog, 2008).
SSCs are reported to be the only dividing somatic cells in the testis; however, two distinct populations of somatic cells were observed dividing near the testis tip. One group, which constituted 69% of phospho-histone-H3-positive somatic cells, appeared to be immediately adjacent to the hub, similar to GSCs. Somatic cells were also observed that were dividing 1-2 cell distances away from the hub. Several scenarios could explain these observations: there are two SSC populations, one which gives rise to hub cells and another that sustains the cyst cell lineage, or there are SSCs that produce both hub cells and a transient amplifying somatic cell population (Voog, 2008).
To distinguish between these possibilities, the heat-shock regime was adjusted to label, on average, only one beta-gal+ somatic cell, and the clones derived from these marked cells were analysed. Thirteen per cent of testes examined contained marked somatic cells adjacent to the hub at 1 day after heat shock, which decreased to 9.5%, 3.3% and 2.8% of testes at 5, 10 and 15 days after heat shock, indicating a half-life for SSCs between 5-10 days. Single, marked somatic cells displaced away from the hub were found initially in 11.3% of testes examined at 1 day after heat shock, but this number decreased to 0.5% by 5 days after heat shock, which is consistent with these cells being transient non-stem-cell clones. In contrast, the number of testes that contained marked hub cells increased from 11.9% at 1 day after heat shock to 25.8%, 24.5% and 18.1% at 5, 10 and 15 days after heat shock, respectively. Clones containing all three cell types were observed in 22% and 14% of testes that contained marked SSCs at 5 and 10 days after heat shock, respectively. From these data it is concluded that multipotent SSCs self-renew and contribute to both hub and cyst cell lineages, whereas dividing cyst cells, which are called cyst progenitor cells (CPCs), expand the pool of cells capable of encapsulating newly divided gonialblasts and maturing spermatogonia to ensure terminal differentiation (Voog, 2008).
To identify factors required for incorporation of cells into the apical hub, SSCs were generated that were mutant for genes expressed in both cell types: DE-cadherin, which is encoded by the shotgun (shg) gene, and the transcriptional repressor Escargot (Esg). DE-cadherin is expressed in cyst cells and is strongly enriched in the hub. SSC clones were generated that were homozygous mutant for either the loss-of-function shgIG29 or amorphic shgIH allele. SSC maintenance and frequency of marked hub cells were assayed at various time points. In this experiment, marked cells and their progeny subsequently become permanently labelled by ubiquitous green fluorescent protein (GFP) expression (Voog, 2008).
Heat-shocked wild-type testes possessed GFP+ GSCs and SSCs, as well as GFP+ hub cells that co-stained with FasIII and DE-cadherin. In contrast to wild type, shg mutant GSC and SSC clones were not maintained, indicating that DE-cadherin has a role in stem cell maintenance in the testis, similar to its role in the ovary, presumably by holding stem cells within the niche and close to self-renewal signals (Voog, 2008).
Marked hub cells were observed in 14%, 35% and 65% of wild-type testes examined at 5, 10 and 15 days after heat shock, respectively. Notably, progeny of DE-cadherin mutant SSCs contributed to the apical hub at a frequency similar to progeny from wild-type SSCs. These data indicate that although DE-cadherin is required for SSC maintenance, it is not absolutely required for mediating the contribution of SSC progeny to the hub (Voog, 2008).
To confirm that shg is not required in hub cells for maintaining the apical hub, RNAi-mediated knockdown of shg expression was carried out in hub cells. A FasIII+ apical hub was detected in 100% of testes from 1-day-old, 10-day-old and 20-day-old males, despite a reduction in DE-cadherin expression in hub cells. Testes collected at 20 days also displayed normal expression of a upd reporter (98%) and contained TJ+ (100%) cells near the apical tip. These data support the findings that DE-cadherin is not absolutely required in hub cells to maintain a functional stem cell niche (Voog, 2008).
However, shg is required in SSCs and early cyst cells for maintaining the apical hub. Knockdown of shg in all SSCs and early cyst cells resulted in a decrease in the number of TJ+ cells in 1-day-old males, consistent with a role for shg in SSC maintenance. Surprisingly, decreased levels of DE-cadherin were also observed in hub cells. In 29% of 15-day-old and 44% of 20-day-old males, the apical hub was severely diminished or lost, as determined by FasIII expression. These data support the model that SSCs act as a source of cells to maintain the apical hub (Voog, 2008).
The transcriptional repressor Escargot is expressed in many tissues, including GSCs, early cyst cells and hub cells in the testis. Males carrying a viable, hypomorphic allele of esg, called shutoff, exhibit loss of apical hub cells during development. Therefore, it is hypothesized that esg may be required for regulating the contribution of SSCs to the hub. Mutant labelled SSCs were generated using two amorphic esg alleles (Voog, 2008).
Unlike progeny from shg mutant SSCs, progeny from esg mutant SSCs did not contribute to the hub at the same frequency as wild-type controls: esgL2 mutant GFP+ hub cells were observed in 5%, 3%, 0% and 4% of testes examined at 1, 5, 10 and 15 days after heat shock, respectively. In instances when esg mutant GFP+ hub cells were observed, normal hub morphology was often severely disrupted. These data suggest that esg regulates either the contribution of SSC progeny to the hub, perhaps by facilitating the cell fate transition between SSC and hub cell, or maintenance of hub cell fate (Voog, 2008).
To explore Esg function further, the agametic oskar (osk) mutant phenotype was used, in which SSCs proliferate and express hub markers, resulting in an apparent expansion of the apical hub. If Esg is required for mediating the transition of somatic cyst cells to the apical hub, it was predicted that the expansion of FasIII+ cells would be blocked in an esg;osk double mutant background. In contrast to the expansion of FasIII+ cells in 82% of osk mutant testes, only 22% of testes from esgshof;osk mutant males showed expansion of FasIII, despite there being clearly more TJ+ somatic cyst cells. These data support previous results and indicate that esg is required for the ability of somatic cells to assume and/or maintain hub cell fate (Voog, 2008).
These findings demonstrating that SSCs can adopt a hub cell fate highlight the dynamic nature of the stem cell-niche relationship and provide a mechanism to regulate the size and function of the stem cell niche in the Drosophila testis. In this model, as somatic cells are displaced from the hub, there is a decline in self-renewal and proliferation potential, which could be reinforced by encapsulation of differentiating germ cells. Interestingly, expansion of the somatic cyst cells as a consequence of germline loss suggests that germ cells exert an anti-proliferative influence that must be overcome in SSCs (Voog, 2008).
A better understanding of how stem cell niches are established and regulated in mammalian systems could facilitate modulation of the niche to enhance transplantation of stem cells in regenerative medicine. Conversely, if an expanded or modified niche accompanies tumour progression or metastasis, then blocking niche maintenance programmes (niche ablation) could be used as an important anti-cancer therapeutic (Voog, 2008).
Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. This study demonstrates that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia (Shields, 2014).
Chickadee, the only Drosophila profilin homolog, is required cell intrinsically for GSC maintenance in the testis. As profilin is a regulator of actin filament polymerization and filamentous actin (F-actin) plays a crucial role in the development and stabilization of cadherin-catenin-mediated cell-cell adhesion, profilin likely maintains attachment of Drosophila male GSCs to the hub through its effect on F-actin, which concentrates at the hub-GSC interface where localized adherens junctions anchor GSCs to hub cells. It is proposed that profilin-dependent stabilization of F-actin at the GSC cortex next to the hub may help localize E-cadherin and APC2 to the junctional region. E-cadherin and APC2 in turn may recruit β-catenin/Armadillo, stabilizing the adherens junctions that attach GSCs to the hub. Chickadee may thus facilitate maintenance of GSCs through a cascade of interactions leading to localization and/or retention of both E-cadherin and β-catenin at the hub-GSC interface (Shields, 2014).
E-cadherin plays a crucial role in maintaining hub-GSC attachment. GSC clones mutant for E-cadherin are not maintained. In addition, germline overexpression of E-cadherin delayed GSC loss in stat-depleted GSCs. The results indicate that profilin function is required in GSCs for proper localization of E-cadherin to the hub-GSC interface. Several studies have shown that the actin cytoskeleton plays a crucial role in assembly and stability of adherens junctions. A favored model in the field is that actin filaments indirectly anchor and reinforce E-cadherin-mediated cell junctions by forming an intracellular scaffold for E-cadherin molecules. Indeed, binding to F-actin stabilized E-cadherin and promoted its clustering. Furthermore, the actin cytoskeleton participates in proper localization of E-cadherin molecules to cell-cell contacts. In chic/profilin mutant GSCs, disruption of actin polymerization at the cell cortex leading to local F-actin disorganization may destabilize E-cadherin and reduce its ability to localize to the GSC-hub junction, form clusters and build adequate adherens junctions (Shields, 2014).
Destabilization of E-cadherin may contribute to the mislocalization of APC2 seen in chic mutant GSCs, as E-cadherin recruits APC2 to cortical sites in GSCs. Raising possibilities of a more direct link, actin filaments have been shown to be required for association of APC2 with adherens junctions in the Drosophila embryo and ovary. Treatment of embryos with actin-depolymerizing drugs resulted in complete delocalization of APC2 from adhesive zones and diffuse APC2 staining throughout the cell. Moreover, in ovaries of chic1320/chic221 females, APC2 was substantially delocalized from the plasma membranes of nurse cells and their ring canals, and increased levels of cytoplasmic APC2 staining were observed. Similarly, this study found that APC2 was delocalized from the hub-GSC interface in larval testes of chic11/chic1320 hypomorphs (Shields, 2014).
In several studies, delocalization of APC2 from junctional membranes correlated with detachment of β-catenin/Armadillo from adherens junctions. APC2 co-localizes with Armadillo and E-cadherin at adherens junctions of Drosophila epithelial cells, nurse cells in Drosophila ovaries and at the hub-GSC interface in Drosophila testes. Disruption of APC2 function resulting in significant reduction of junctional APC2 was accompanied by delocalization of junctional Armadillo and increased levels of free cytoplasmic Armadillo in embryonic epithelial cells and ovaries. In a previous study, which used chic1320/chic221 strong loss-of-function mutants, the delocalizing effect on junctional Armadillo was variable, presumably due to incomplete penetrance of chic mutant effects. Although this study did not observe significant disruption in Armadillo staining along the hub-GSC interface of testes from chic hypomorphs, this may be due to incomplete penetrance. In addition, the Armadillo protein detected could be localized to the cortex of hub cells rather than GSCs (Shields, 2014).
The finding that germline specific overexpression of APC2 in chic11/chic1320 hypomorphs partially rescued GSC loss is consistent with a previously proposed model that actin filaments shuttle APC2 to adherens junctions and APC2 in turn recruits cytoplasmic Armadillo to junctional membranes, reinforcing the adherens junctions. It is possible that in chic11/chic1320 hypomorphs, residual actin filaments associated with adherens junctions between the hub and GSCs are sufficient to shuttle the increased amounts of cytoplasmic APC2 to adherens junctions. This APC2 may in turn recruit free cytoplasmic Armadillo to the hub-GSC interface, locally stabilizing the adherens junctions and anchoring GSCs to their niche. Notably, however, germline specific overexpression of APC2 in testes of strong loss-of-function chic1320/chic221 mutants failed to rescue GSC loss. Thus either, adequate levels of actin filament polymerization may be required for the proposed translocation of junctional proteins to the plasma membrane, or APC2 function/localization may not be the only or even the major cell-autonomous target of profilin function important for maintaining GSCs. Indeed, loss of APC2 function did not lead to GSC loss. It is suggested that the localized cortical F-actin underlying adherens junctions at the GSC-hub interface, best candidate for the most direct target of chic function, strongly stabilizes adherens junctions between GSCs and the hub, with high levels of cortical APC2 able to in part make up for weak chic function by also stabilizing adherens junctions (Shields, 2014).
Maintenance of hub-GSC attachment appears to be a key role of STAT in GSCs. The finding that STAT binds to a site near the upstream promoter of the chic gene raises the possibility that STAT might foster GSC attachment to the hub in part by ensuring high levels of transcription of profilin in GSCs. However, activation of STAT is clearly not the only regulatory influence on profilin expression as profilin is an essential gene expressed in many cell types, including those in which STAT is not active or detected. It is likely that transcription factors other than STAT turn on profilin expression in many cell types and that STAT acts along with other regulators to reinforce profilin expression in GSCs and CySCs. Conversely, overexpression of profilin was not sufficient to re-establish attachment of stat-depleted GSCs, suggesting that STAT probably regulates a number of genes to ensure that GSCs remain within the stem cell niche (Shields, 2014).
Loss of chic function in somatic cyst cells impaired the ability of cyst cells to build and/or maintain the cytoplasmic extensions through which they embrace and enclose spermatogonial cysts. Two somatic cyst cells normally surround each gonialblast and enclose its mitotic and meiotic progeny throughout Drosophila spermatogenesis. The cyst cells co-differentiate with the germ cells they enclose. Several lines of evidence support the model that either the ability of somatic cyst cells to enclose germ cells or their ability to send signals to adjacent germ cells is important to restrict proliferation and promote differentiation of germ cells. In either case, activation of EGFR in cyst cells is required for cyst cells to enclose germ cells and/or send the signals for germ cells to differentiate. The similarities in phenotype between loss of chic function and loss of EGFR activation in somatic cyst cells raise the possibility that chic/profilin may act downstream of activated EGFR to modulate the actin cytoskeleton for the remodeling of cyst cells to form or maintain the cytoplasmic extensions that enclose germ cells. Indeed, activated EGFR is known in other systems to tyrosine phosphorylate phospholipase C-γ1 (PLC-γ1), a soluble enzyme in quiescent cells like daughter cyst cells, activating it to catalyze hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which binds profilin protein with high affinity, which inhibits the interaction between profilin and actin. The hydrolysis of PIP2 by activated PLC-γ1 results in localized release of profilin and other actin-binding proteins, enabling them to interact with actin and participate in cytoskeletal rearrangement and membrane protrusion. Thus, based on biochemical analysis in other systems, a link between EGFR activation and profilin leading to local remodeling of the actin cytoskeleton is plausible in somatic cyst cells, although it remains to be directly tested (Shields, 2014).
Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). This study used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. It is proposed that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo (Mavrakis, 2008).
To study the organization of the PM and the spatiotemporal dynamics of membrane components in living Drosophila embryos, transgenic animals were generated expressing different PM proteins tagged with Cerulean or Venus fluorescent proteins. The proteins were selected because they have different modes of membrane attachment and potentially different PM distributions. They included: (1) Venus fused to the first 20 amino acids of growth-associated protein 43 (GAP43), which contain a dual palmitoylation signal that tightly anchors the protein to the inner leaflet of the PM, (2) Cerulean fused to the pleckstrin-homology domain of phospholipase C delta 1, PH(PLCδ1), which binds specifically to the phosphoinositide PI(4,5)P2, and (3) Venus fused to full-length Toll receptor, a type I transmembrane protein that is required for dorsal-ventral embryonic polarity (Mavrakis, 2008).
This study provides evidence that the plasma membrane of the fly syncytial blastoderm exhibits a polarized, epithelial-like organization prior to cellularization. Previously, it was thought that the PM of the blastoderm had no specialized organization prior to the formation of cell boundaries at cellularization. The results show that despite the absence of cell boundaries, the PM of the syncytial blastoderm has apical- and basolateral-like domains surrounding individual cortical nuclei and that PM proteins do not exchange between PM regions surrounding adjacent nuclei. This organization is maintained throughout syncytial mitotic division cycles and is dependent on an intact F-actin network (Mavrakis, 2008).
Support for these conclusions came from live imaging and fluorescent highlighting experiments in living embryos. Using a variety of membrane markers, two distinct PM regions were distinguished. One region was above individual nuclei and had apical-like characteristics, including the presence of microvilli and an enrichment in PI(4,5)P2, a key determinant of apical PM biogenesis, as well as in GAP43, a protein that localizes to raft-like membranes, which typically compose apical PM surfaces in epithelial cells. The second PM region was lateral to nuclei, and was enriched in markers typically associated with basolateral membranes and junctions, including the cell-cell adhesion molecule E-cadherin, the multi-PDZ domain scaffolding protein DPatj. FRAP experiments showed that the molecules could freely diffuse in the PM domains surrounding individual nuclei but did not diffuse outside them, suggesting the presence of a diffusion barrier between the domains during interphase. Moreover, optical pulse-chase experiments showed that these components did not diffuse outside PM domains surrounding mitotic units throughout the time period of syncytial divisions. Thus, during mitosis, the polarized organization and restricted diffusion pattern of proteins in the PM did not change. Finally, the requirement of an intact F-actin network was supported by drug-induced actin depolymerization, which disrupted PM association of DPatj and Peanut and abolished the restricted diffusion pattern in the PM (Mavrakis, 2008).
The finding that the PM of the syncytial blastoderm is organized as a pseudoepithelium prior to cellularization has several important implications for understanding many aspects of embryo development. First, it directly impacts on how dorsal-ventral and terminal patterning are set up prior to cellularization. These are dependent on Toll and Torso membrane receptors. Toll is distributed uniformly along the syncytial PM, but is activated only ventrally. Similarly, Torso is uniformly expressed along the surface membrane of early embryos, but its activation occurs only at the anterior and posterior poles. Given that membrane receptors have the capacity to diffuse across the PM, it has been unclear why the activation zones of these receptors do not spread widely across the PM. The results revealing the compartmentalized character of the PM during interphase and syncytial nuclear divisions now provide a potential answer. Receptors diffuse locally within the PM surrounding a particular nucleus, but they do not diffuse to PM regions associated with other nuclei. Consequently, activation zones of receptors (set up by the localized spatial signal of ligands) do not spread, allowing robust downstream signaling events in particular regions of the embryo. This possibility is supported by the spreading of the Dorsal gradient to more anterior and posterior regions in embryos treated with latA. LatA-induced actin depolymerization abolished the confined diffusion pattern in the PM suggesting that an intact actin network is likely to be important for containing activated Toll diffusion and thus maintaining a robust downstream Dorsal gradient (Mavrakis, 2008).
The molecular basis for the compartmentalized diffusion in the PM of the syncytial embryo appears to be due to the presence of bona fide diffusion barriers in the PM regions directly between adjacent nuclei. The finding that septins and components of junctions are specifically enriched in this PM region raises the possibility that these molecules together with other cytoskeletal components organize a barrier to diffusion in the plane of the PM in a way similar either to the organization of septin rings at the yeast bud neck or of adherens junctions in epithelial cells. Moreover, the loss of PM association of DPatj and Peanut, as well as the abolishment of the restricted diffusion pattern in latA-treated embryos, suggest that an intact F-actin network is required both to localize and/or maintain septins and junctional components to specialized PM regions and to contain diffusion of proteins in PM units around individual syncytial nuclei. An intact F-actin network was recently shown to be required for compartmentalizing furrow canals during cellularization further supporting that F-actin organizes lateral diffusion of proteins in the PM. Future studies will need to genetically dissect the molecular machineries involved in organizing such diffusion barriers (Mavrakis, 2008).
A second implication of the observed PM dynamics during syncytial mitoses relates to the machinery driving PM invagination. It was found that the PM was organized into highly convoluted microvillous membrane buds over interphase nuclei and these flattened out as soon as nuclei entered mitosis before reorganizing again into microvillous buds upon re-entry into the next interphase. Furthermore, the rate at which PM invaginated (~1.5-2 μm/min) was twice as fast as during the fast phase of cellularization, which involves de novo membrane delivery. Although endocytosis was recently shown to accompany metaphase furrow ingression, the current observations support a mechanism for PM invagination in mitosis that involves contractile machinery which transiently redistributes PM from microvilli caps into transient furrows surrounding mitotic units rather than an internal membrane source (Mavrakis, 2008).
A final implication of these findings relates to cellularization, which produces the primary epithelial cells of the embryo. Polarization of the invaginating PM during cellularization has been reported, and it is during cellularization that PM polarity is first thought to be achieved in early fly embryogenesis. Because the data demonstrate that the PM is already polarized prior to cellularization, it is likely that the embryo uses this organization to initiate and organize the cellularization process. Consistent with this, it was found that the junctional proteins E-cadherin and DPatj, the septin protein Peanut, and Toll are all highly enriched in the PM at sites between adjacent nuclei during syncytial interphases, which reflects the PM organization between nuclei right at the onset of cellularization (first few minutes of interphase 14). Indeed, these are precisely the PM sites that become further differentiated within the first 5 min into cellularization, with the formation of an invaginating membrane front that contains Peanut and DPatj, basal adherens junctions directly adjacent to the invaginating front that contain E-cadherin, and the extension of the lateral membranes that are positive for Toll. The epithelial polarization occurring during cellularization is thus already reflected in the organization of the syncytial blastoderm PM (Mavrakis, 2008).
In summary, these findings that the syncytial blastoderm PM exhibits an epithelial-like polarization prior to cellularization, and that distinct PM domains do not significantly exchange membrane components, point to an as yet unexplored mechanism for how the embryo maintains and generates morphogen gradients at this stage. By preventing activation zones of membrane receptors on the PM from spreading, robust downstream signaling events within the cytoplasm and nuclei of the embryo can be established. This mechanism would work in conjunction with nuclear-cytoplasmic shuttling of transcription factors, and a compartmentalized secretory pathway, to generate the dorsal-ventral and terminal patterning systems of the blastoderm fly embryo (Mavrakis, 2008).
Organs develop distinctive morphologies to fulfill their unique functions. Drosophila embryonic gonads were used as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. A membrane GFP marker was developed to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. It is proposed that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction (Sano, 2012).
SGPs and PGCs display dramatic changes as they progress from cell aggregates to the well-compacted embryonic gonads. In the mature gonad, PGCs are embedded in a mesh network of SGP cytoplasmic extensions, and the interaction between these cells is enhanced by SGP-driven gonad compaction. The compaction process is important not only in maintaining the structural integrity of the gonad but also in ensuring proper differentiation of germline stem cells, which requires the intimate association of PGCs and the niche derived from anterior SGPs. This study found that Ena, an actin regulator, is required for gonad compaction. Ena acts in the soma, and loss of ena alters SGP cell shape and orientation, and intracellular distribution of DE-cad. Mathematical simulation suggests that Ena has an important role in regulating the cadherin-dependent cell adhesive forces necessary for proper organ morphogenesis (Sano, 2012).
Detailed analysis of SGP morphology during gonad formation revealed that gonad compaction relies on coordinated changes in SGP cell shape and orientation. Anterior SGPs turn less than those located in the middle or posterior pole, which may contribute to the overall anterior movement of the gonad primordium from its original position at PS10-12 to its final location at PS10-11. Consistent with their failure to form a compact gonad, the angle of ena mutant SGPs is smaller with respect to the gonad AP axis (Sano, 2012).
It was noticed that the ena mutation affects SGP cell shape and axis orientation differently in different regions of the gonad. Defects in cell shape changes were more severe at the anterior than in the posterior, while cell orientation was disturbed more severely in the middle and posterior SGPs. Since ena is detected uniformly in all SGPs, these phenotypic differences are likely due to internal differences in the SGPs. In the early stages of development, SGPs are specified as three clusters in PS10, 11, and 12 in the mesoderm. The homeobox gene, abd-A, is expressed in PS10 through 12, while abdominal-B (abd-B) is only expressed in PS11 and 12 at the coalescence stage. Although the ultimate fate of individual SGPs from each parasegment remains unknown, differential expression of abd-A and abd-B could account for the different cellular behaviors (Sano, 2012).
It is well established that Ena family proteins promote actin filament elongation. Therefore, it is likely that Ena is involved in DE-cadherin dynamics through regulation of the local actin morphology in SGPs. Ena translocation to the inner surface of SGPs might accelerate actin filament bundling at the inner surface. DE-cad is known to associate with bundled actin filaments and this may contribute to its accumulation at the inner surface of SGPs. Mechanical force could also be involved in DE-cad localization. In vitro analysis has demonstrated the force-dependent recruitment of the actin-binding protein, Vinculin, by α-Catenin. Adhesion by E-cad to the adjacent cell stretches the membrane, with the apparent transmission of the stretching force to α-Catenin resulting in the recruitment of Vinculin and actin filaments. This mechanism could also act at the contact surface between SGPs, thus stabilizing DE-cad (Sano, 2012).
Recent studies have shown that cellular pattern formation during morphogenesis is coordinated via the localization and/or activity of force-generating molecular machinery, such as cell adhesion molecules and Myosin. Thus, the altered DE-cad distribution in ena mutants prompted an examination of whether ena controls the force balance in gonadal cells by regulating DE-cad. Since ena expression and function are necessary in the soma, and PGCs are dispensable for gonad compaction, it is reasonable to focus on SGPs to determine the parameters controlling these forces by numerical simulation. in silico analysis showed that gonad compaction is promoted by increased adhesive force on the inner surface. This is consistent with the DE-cad relocation observed in vivo. Larger adhesive forces between SGPs increase SGP-SGP adhesion surfaces leading to SGP rearrangement and incorporation to form a spherical gonad. It was also shown that a larger contraction force along the outer surface (between the SGP and surrounding non-gonadal environment) resulted in increased gonad compaction; however, no significant Myosin II accumulation at the outer membrane of SGPs was detected. One possibility is that Myosin activity, instead of localization, is increased at the outer membrane. Alternatively, Myosin II could act in the SGP cytoplasmic extensions to contract them, thereby generating the inward force required for gonad compaction. However, no overt changes were observed in the cellular protrusions in ena mutants, making this alternative less likely (Sano, 2012).
A previous study found that robo genes are required for gonad compaction and Robo2 was localized at contact sites between SGPs. The Robo receptor family is reported to be involved in homophilic and heterophilic adhesion and repulsion. Indeed, Ena has been shown to bind to Robo, suggesting that the observed effects of Robo on gonad morphogenesis could be mediated by Ena. Ena's function appears, however, primarily associated with the morphogenetic movements and cell shape changes observed during gonad formation, as ena mutants, in contrast to robo mutants, do not affect PGC ensheathment. Future quantitative measurement of force dynamics and force-generating molecular machinery, coupled with live observation in specific genetic backgrounds, will further clarify the mechanics of gonad compaction (Sano, 2012).
Polarised tissue elongation during morphogenesis involves cells within epithelial sheets or tubes making and breaking intercellular contacts in an oriented manner. Growing evidence suggests that cell adhesion can be modulated by endocytic trafficking of E-cadherin (E-cad), but how this process can be polarised within individual cells is poorly understood. The Frizzled (Fz)-dependent core planar polarity pathway is a major regulator of polarised cell rearrangements in processes such as gastrulation, and has also been implicated in regulation of cell adhesion through trafficking of E-cad; however, it is not known how these functions are integrated. This study reports a novel role for the core planar polarity pathway in promoting cell intercalation during tracheal tube morphogenesis in Drosophila embryogenesis, and evidence is presented that this is due to regulation of turnover and levels of junctional E-cad by the guanine exchange factor RhoGEF2. Furthermore, it was shown that core pathway activity leads to planar-polarised recruitment of RhoGEF2 and E-cad turnover in the epidermis of both the embryonic germband and the pupal wing. This study thus reveals a general mechanism by which the core planar polarity pathway can promote polarised cell rearrangements (Warrington, 2013).
Looking in three different tissues, this study found that core planar polarity pathway activity promotes E-cad turnover from junctions, most likely via local recruitment and regulation of RhoGEF2 and RhoA activity. In general terms, it is believed that local assembly or disassembly of adherens junctions through trafficking of E-cad is likely to be important for polarised tissue rearrangement; however, few specific contexts in which this occurs have been identified (Warrington, 2013).
One process in which regulation of E-cad turnover is strongly linked to cell intercalation is elongation of branches in the Drosophila embryonic tracheal system. Loss of core pathway function and also reduction of RhoGEF2 and RhoA activity give a similar phenotype to blocking endocytosis in this tissue, resulting in increases in both overall levels of and the stable fraction of E-cad at junctions, and a delay in cell intercalation. Consistent with the increase in E-cad levels being the cause of the intercalation defect in core pathway backgrounds, this phenotype can be suppressed by lowering E-cad gene dosage. It is speculated that core planar polarity proteins might transiently show polarised distribution or activity in this context, thus selectively weakening junctions and allowing cells to slide over one another. However, consistent with previous studies, this study failed to detect such asymmetry. The possibility cannot therefore be ruled out that core pathway activity is uniform within cells in this tissue, and only plays a role in general modulation of E-cad trafficking (Warrington, 2013).
In the pupal wing, the core pathway has already been linked to regulation of E-cad trafficking, and evidence has been presented that this promotes junctional remodelling that gives rise to a regular hexagonal arrangement of the cells. The exact mechanism by which the core pathway modulates E-cad trafficking was not defined, although the observation that Sec5 is recruited to proximodistal junctions suggested that there might be a role for local exocytosis of E-cad. Looking at a stage shortly after junctional remodelling, when the core proteins are strongly asymmetrically distributed, planar-polarised localisation of RhoGEF2 to proximodistal junctions is observed, but also a decrease in overall levels and the stable fraction of E-cad in this position. This appears to rule out a role for increased E-cad exocytosis on proximodistal junctions. Interestingly, although Sec5 is best characterised as a component of the exocyst, it has also been implicated in endocytosis in the Drosophila oocyte and perhaps this is also true in the wing. It is not clear how planar-polarised E-cad trafficking would contribute to formation of a regular hexagonal array of cells, as removing E-cad from proximodistal distal junctions might be expected to cause shrinkage of these junctions at the expense of anteroposterior junctions. However, during the peak period of junctional rearrangement (from ~18 hours of pupal life), the planar-polarised asymmetric distribution of the core proteins is largely lost, and so it might be that during the crucial stage of morphogenesis the core pathway promotes relatively uniform endocytosis of E-cad (Warrington, 2013).
The observation of a role for the core pathway in modulating E-cad turnover in the epidermis of the embryonic germband is particularly intriguing, as loss of core pathway activity does not result in a defect in embryonic germband extension, even though a planar-polarised distribution of E-cad has been implicated as a key mechanism in promoting cell intercalation in this context. It is speculated that planar polarisation of E-cad might be only one of a number of mechanisms that operate redundantly during the crucial developmental event of germband extension. Among other mechanisms reported are localised actomyosin contraction at vertical junctions, inhibition of Bazooka localisation on vertical junctions by local Rho kinase (Rok) activity and alteration of Arm (β-catenin) dynamics on vertical junctions by localised activity of the Abl kinase. Interestingly, it was found that loss of core pathway activity also abolishes Zipper and Bazooka asymmetry, but not Arm asymmetry (Abl or Rok asymmetry was not examined in this study). Additionally, a planar-polarised distribution of activated Src kinase to horizontal junctions was observed. Although Src kinase is a known modulator of E-cad trafficking in the Drosophila embryo, the significance of its planar-polarised distribution is unclear, as loss of core pathway function did not affect the distribution of Src, but did block the planar-polarised distribution of E-cad (Warrington, 2013).
Another context in which the core pathway might modulate E-cad turnover is during ommatidial rotation in the developing Drosophila eye, in which possible involvement of both RhoA and the kinase Nemo have been reported (Warrington, 2013).
In summary, this study has presented evidence that the core planar polarity pathway acts to locally promote E-cad endocytosis via local recruitment of RhoGEF2 and activation of RhoA activity. This represents a mechanism by which the core pathway can promote planar-polarised cell rearrangements (Warrington, 2013).
Fibroblast growth factor (FGF)-dependent epithelial-mesenchymal transitions and cell migration contribute to the establishment of germ layers in vertebrates and other animals, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been provided for any system. The establishment of the Drosophila mesoderm layer from an epithelial primordium involves a transition to a mesenchymal state and the dispersal of cells away from the site of internalisation in a FGF-dependent fashion. This study shows that FGF plays multiple roles at successive stages of mesoderm morphogenesis in Drosophila. The two FGF ligands, Pyr and Ths, have multiple, partially overlapping functions in directing this morphogenetic behaviour. FGF signaling is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions. These protrusions extend deep into the underlying ectoderm epithelium and are associated with high levels of E-cadherin at the germ layer interface. Finally, FGF directs distinct hitherto unrecognised and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to a dorsally directed movement across its surface. Both movements are important for layer formation, and evidence is presented suggesting that they are controlled by genetically distinct mechanisms (Clark, 2011).
The rapid flattening of the mesoderm onto the ectoderm surface has been attributed to decreased adhesion between mesoderm cells as a result of the mitotic division that follows internalisation. An alternative view proposes that the mesoderm actively contacts the ectoderm involving mesoderm cell protrusions. This study now finds that the mesoderm extends actin-rich protrusions towards the ectoderm as the tissue flattens. The formation of these protrusions depends on FGF signalling, which suggests a role for the FGF pathway in controlling the dynamics of the actin cytoskeleton (Clark, 2011).
Mesoderm flattening occurs by a zippering motion, with progressive attachments that commence in the most ventral region and propagate to more dorsolateral positions. It is proposed that the region in which protrusions are formed expands dorsally, because flattening of ventral parts of the mesoderm exposes more dorsally located cells to the influence of FGF expressed by the neuroectoderm. This propagation model of mesoderm flattening helps to explain on a cellular level how cells from defined initial positions follow apparently stereotypical paths. Such a mechanism would provide for an orderly association of the germ layers, ensuring that mesoderm cells are symmetrically distributed about the ventral midline (Clark, 2011).
It has been proposed that mesoderm spreading may be driven by differential adhesion. A propensity for the mesoderm to maximise contact with the ectoderm would follow from mesoderm cells that exhibit a higher affinity for the ectoderm than for each other. Consistent with earlier studies, this study found that although E-cadherin transcription is repressed by Snail in the mesoderm, maternal E-cadherin protein levels do not rapidly decrease upon EMT. During EMT, E-cadherin distributes over the whole cell surface of the mesoderm cells. As contact with the ectoderm is made, E-cadherin accumulates at the germ layer interface, including the sites where mesodermal protrusions penetrate the ectodermal layer. E-cadherin mutants exhibit mild defects in dorsal mesoderm morphogenesis, but the function of E-cadherin in differential adhesion or in promoting mesoderm-ectoderm attachment and spreading is not yet understood. To address this issue, it will be necessary to establish a system for tissue-specific conditional interference with E-cadherin (Clark, 2011).
It is also possible that molecules other than E-cadherin mediate adhesion between the ectoderm and the mesoderm germ layer. Although the prime candidate for this mechanism is integrin-mediated adhesion, evidence was found for a role of integrins in mesoderm spreading. The model is therefore favored that E-cadherin has a major role in establishing adhesion between the germ layers (Clark, 2011).
FGF signalling contributes to a switch in the state of E-cadherin leading to the redistribution of polarised E-cadherin during EMT in the invaginated mesoderm cells. A similar switch in E-cadherin function occurs during border cell migration in Drosophila oogenesis. Although the cytoplasmic domain of E-cadherin contains a conserved function necessary for cell migration, it is unclear how the E-cadherin in migrating cell collectives is linked to the cytoskeleton to allow it to transmit the forces required for movement. The cell contacts involved in these movements need not be stable adherens junctions, but are perhaps rather dynamic interactions. This study identifies Cdc42 as an important determinant of both protrusion formation and E-cadherin accumulation at the ectoderm/mesoderm interface. Further studies will have to address the mechanisms by which Cdc42 is controlled and functions upstream of E-cadherin localisation (Clark, 2011).
This study has revealed that dorsal edge cells undergo successive changes in protrusive behaviour. The biphasic movement of DECs and repolarisation of protrusive behaviour correlate in timing with the switch in pyr mRNA distribution to a more restricted expression in the dorsal ectoderm. This study shows that Pyr is indeed required for the normal migratory behaviour of cells at the dorsal edge. It is proposed that as DECs migrate dorsally, opportunities are created in their wake for the intercalation of inner mesoderm cells into the monolayer more ventrally (Clark, 2011).
The relevance of the radial protrusive activity for mesoderm spreading is less easy to understand. Pyr and Ths are both required for E-cadherin redistribution and radial protrusion formation during mesoderm flattening. The patterns of the paths derived by tracking all mesodermal cells have hinted at intercalation as an important mechanism of mesoderm layer formation. The radial protrusive activity indicates a continuous attraction of mesoderm protrusions towards the ectoderm. It is proposed that a main function of FGF signalling on a cellular level is to direct protrusive activity into two overall directions: dorsal and radial. Earlier studies have shown that dorsal protrusions depend on the Rac pathway, whereas this study shows that radial protrusions are particularly sensitive to loss of Cdc42 function. These results suggest that FGF signals might be differentially transduced within the migrating collective or that radial protrusive activity uses distinct molecular pathways (Clark, 2011).
Based on the evidence presented, it is proposed that FGF signalling performs three key functions in controlling mesoderm cell behaviour: (1) FGF triggers actin-dependent protrusive activity during flattening; (2) FGF induces modulation of E-cadherin distribution during EMT; (3) FGF acts as an attractant for dorsal migration. Therefore, the key cellular processes that depend on FGF are the remodelling of E-cadherin adhesions and the guidance of directional protrusive activity. Although the molecular details of the signalling pathways remain to be discovered, these data suggest that distinct small GTPase pathways, such as Cdc42 and Rac, play crucial roles in determining the specificity of the FGF signalling responses that direct cell behaviours during mesoderm layer formation (Clark, 2011).
Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. This study shows that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell-GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers (Hsu, 2009).
The stem cell microenvironment (niche) controls stem cells, and niche aging leads to stem cell decline. The Drosophila germline stem cell (GSC) niche includes terminal
filament cells, cap cells, and escort stem cells, and GSC fate and
activity require direct contact with cap cells and exposure to
niche-derived signals. GSCs also respond to systemic signals,
such as Drosophila insulin-like peptides (DILPs), which
directly modulate their proliferation. Increased age leads to
decreased niche size and signaling and GSC loss. The molecular basis for age-dependent changes in the niche, however, remains poorly understood (Hsu, 2009).
Because diet influences aging, its effects on GSC maintenance were examined, exploiting the fact that GSCs can be unambiguously identified by their anteriorly anchored fusome (a membranous cytoskeletal structure) and by their juxtaposition to cap cells. A decrease was observed in GSC numbers in well-fed females over time. In females on a poor diet, however, the rate of GSC loss was significantly increased (Hsu, 2009).
Insulin secretion and signaling respond to diet and
diminish in aging humans. Using a phosphoinositide
3-kinase reporter, reduced insulin signaling was found in
older ovaries. To address if GSC maintenance requires
insulin signaling, GSC numbers were measured in Drosophila
insulin receptor (dinr) mutants. The
dinr339/dinrE19 females contain slightly fewer GSCs at eclosion and lose them significantly faster than controls. GSC death was not observed in dinr339/dinrE19 or control
germaria, suggesting that GSC loss results from differentiation (Hsu, 2009).
The chico1 homozygotes, which lack insulin receptor substrate, a
major insulin pathway component, also show increased GSC loss. Thus, insulin signaling controls GSC maintenance. Next, whether DILP expression in germarial somatic cells
could counteract the wild-type age-dependent GSC loss was tested. The c587-GAL4 driver was used to express a UAS-dilp2 transgene, encoding the DILP most closely
related to human insulin, and thereby increase the local
levels of insulin-like signals. GSC loss on rich and poor diets was
significantly suppressed by DILP2 overexpression, although this
was less pronounced in 4-week-old females on a poor diet. The less effective rescue on a poor diet could potentially be attributable to lower expression of the c587-GAL4
driver, to the actions of additional diet-dependent signals, or to
a combination thereof. Nevertheless, these results suggest that
the normal GSC loss observed in wild-type females as their age
increases results largely from reduced insulin signaling (Hsu, 2009).
DILPs control GSC division directly, leading to a cell-autonomous
dinr requirement. It was therefore asked whether
dinr is required within GSCs for their maintenance. In genetic
mosaics, homozygous dinr339 or dinrE19 GSCs are not lost at a higher rate than control GSCs, demonstrating that
DILPs do not promote GSC maintenance directly (Hsu, 2009).
It was next hypothesized that insulin signaling may regulate GSC
fate via the niche. Indeed, expression of wild-type dinr in somatic
cells of dinr339/dinrE19 germaria rescued GSC loss. To
examine dinr339/dinrE19 niche structure, terminal
filament and cap cells were counted. Terminal filament cell
numbers in dinr339/dinrE19 and control females are similar. In contrast, dinr339/dinrE19 females eclose with fewer cap cells and also lose them faster over time, suggesting that
insulin signaling controls cap cell number during development
and adulthood. Moreover, DILP2 overexpression suppresses the
wild-type age-dependent cap cell number decrease. It is
concluded that DILPs control GSC niche size and that the
reduced cap cell numbers observed with increased female age at
least in part reflect low insulin signaling levels (Hsu, 2009).
It was next asked whether DILPs control cap cell number
directly. In mosaic germaria containing β-gal-negative dinr339 or
control cap cells, the distribution
(and average number) of β-gal-negative cap cells was indistinguishable, indicating that dinr does not control cap cell number cell autonomously. It is possible that a second cell type, such as terminal filament cells, produces an intermediate
factor; alternatively, cap cells themselves may control their own
maintenance via paracrine signaling (Hsu, 2009).
Notch signaling controls cap cell number during niche formation
and in adults. Notch hyperactivation during development
forms ectopic cap cells, leading to excess GSCs. Conversely,
defective Notch signaling reduces niche size and GSC
number. Notch activation is strongly detected in larval terminal
filament and cap cells and is also detected in adult cap cells. Notch signaling was examined in dinr mutants using the E(spl)mβ-CD2 reporter. Every control
germarium had strong CD2 expression in both terminal
filament and cap cells. In contrast, CD2 levels were severely
reduced in dinr339/dinrE19 germaria, indicating that
insulin signaling controls Notch activation in the niche (Hsu, 2009).
It was next asked if the reduced cap cell number in dinr mutants
was attributable to impaired Notch signaling. Weak
hypomorphic dinrE19/dinr353 females have no reduction in GSC or cap cell number. Similarly, Notch
heterozygotes (half the Notch dosage) have normal GSC and cap
cell numbers. In contrast, dinrE19/dinr353 females heterozygous for Notch have significantly reduced GSC and cap cell
numbers. A decrease in small cap cell number has been reported
for Notch heterozygotes; this discrepancy may reflect slightly reduced insulin signaling in the latter study attributable to diet (Hsu, 2009).
To determine if Notch signaling is sufficient to rescue dinr defects, an activated form of Notch was expressed in the somatic cells of dinr339/dinrE19 germaria, and the GSC and cap cell loss phenotypes were rescued. These results and the genetic interaction between dinr and Notch are consistent with the insulin pathway acting upstream or in parallel to Notch. Nevertheless, the reduced Notch reporter levels in dinr
mutants favor the model that insulin signaling leads to Notch activation, thereby controlling cap cell number and, indirectly, GSC maintenance (Hsu, 2009).
GSCs and terminal filament cells express the Delta ligand for Notch, and removal of Delta function from GSCs has been reported to affect niche activity. It was reasoned that dinr could be required in GSCs, terminal filament cells, the cap cell population, or a combination thereof to control Delta production and Notch activation. dinr mosaic germaria were examined in which all GSCs were dinr339 homozygous, and the number of cap cells in those germaria was indistinguishable from control numbers, suggesting that dinr is not required in GSCs for Notch signaling. DILPs may instead regulate Delta within terminal filament or cap cells or, alternatively, act via other intermediate signals to regulate Notch activation within the niche (Hsu, 2009).
Cap cell and GSC numbers correlate. Indeed, in germaria containing control β-gal-negative cap cells (control C1), total cap cell and GSC numbers are roughly proportional. Remarkably, despite similar cap cell numbers, a significant fraction of germaria in which dinr mutant cap cells are present contains fewer GSCs relative to control C1 or C2 (i.e., germaria without cap cell clones from dinr mosaics). Thus, although dinr does not control cap cell number per se autonomously, it is required within cap cells either for the optimal production and/or secretion of a GSC maintenance
factor(s) or to promote GSC attachment (Hsu, 2009).
Niche-derived bone morphogenetic protein (BMP) signals directly stimulate GSCs to repress differentiation. To test if insulin signaling controls BMP pathway activation in GSCs, the Dad-lacZ reporter was used. Dad-lacZ levels in dinr339/dinrE19 and control females are indistinguishable, showing that dinr does not control BMP signaling. Insulin signaling in cap cells must therefore control another GSC maintenance signal and/or the cap cell-GSC association (Hsu, 2009).
To investigate if dinr controls the physical interaction between cap cells and GSCs, the percentage of dinr339 versus control cap cells directly contacting GSCs was measure in mosaic germaria. Indeed, 21% of dinr339 cap cells contact GSCs, compared with 50% of control cap cells, indicating that dinr339 cap cells have significantly reduced attachment to GSCs. These results suggest that insulin signaling in cap cells controls their association with GSCs. Alternatively, insulin signaling may regulate the production of a short-range GSC maintenance signal, such that only GSCs in contact with dinr mutant cap cells are affected (Hsu, 2009).
E-cadherin-mediated adhesion between cap cells and GSCs
is required for retaining GSCs in the niche. Therefore E-cadherin levels were measured at the GSC-cap cell junction. In controls, it was found that E-cadherin levels vary with changes in the fusome, a membranous cytoskeletal structure. When the fusome is round, its predominant morphology, there is a higher intensity of E-cadherin at the junction, although when the fusome is elongated, the intensity is lower. The intensity of E-cadherin at the junction of cap cells with GSCs displaying elongated fusomes in dinr339/dinrE19 mutants is similar to that of control. In contrast, the round fusome GSC-cap cell junctions contain significantly lower E-cadherin levels in dinr mutants than in controls. These results suggest that insulin signaling influences E-cadherin levels at the GSC-cap cell junction and may explain the age-dependent E-cadherin reduction that contributes to GSC loss (Hsu, 2009).
These studies demonstrate that systemic insulin-like signals integrate inputs from diet and age to regulate GSC maintenance via the niche. Specifically, it is proposed that DILPs control cap cell number via Notch and also E-cadherin- mediated GSC retention within the niche. Because diet and insulin signaling control GSC proliferation, it is also likely that the proliferation decline reported in older females results from reduced insulin signaling. These results also provide insights into recent findings that systemic factors from young mice can restore Notch activation and skeletal muscle progenitor proliferation and regenerative capacity to old mice in heterochronic parabiotic pairings. Finally, the results are intriguing in light of the well-established connection between low insulin signaling, restricted diet, and extended lifespan and of studies in C. elegans suggesting that GSCs may have a negative effect on longevity. It is conceivable that excessive stem cell activity in general is deleterious and that slight decreases in stem cell number or activity with age as a result of reduced insulin signaling may actually promote longevity (Hsu, 2009).
When single cells or tissues are injured, the wound must be repaired quickly in order to prevent cell death, loss of tissue integrity, and invasion by microorganisms. Drosophila is a genetically tractable model to dissect the mechanisms of single-cell wound repair. By analyzing the expression and the effects of perturbations of actin, myosin, microtubules, E-cadherin, and the plasma membrane, three distinct phases in the repair process were defined (expansion, contraction, and closure), and specific components required during each phase were identified. Specifically, plasma membrane mobilization and assembly of a contractile actomyosin ring are required for this process. In addition, E-cadherin accumulates at the wound edge, and wound expansion is excessive in E-cadherin mutants, suggesting a role for E-cadherin in anchoring the actomyosin ring to the plasma membrane. The results show that single-cell wound repair requires specific spatial and temporal cytoskeleton responses with distinct components and mechanisms required at different stages of the process (Abreu-Blanco, 2011).
By combining high resolution imaging and multiple fluorescence markers in both wild-type and mutant embryos, the Drosophila single-cell repair model facilitates the systematic analysis of specific components and provides new mechanistic insights on how these components are specifically recruited and dynamically localized during the healing process. Although basic descriptions of single-cell wound repair are in place from studies in different wound models, a number of fundamental questions remain concerning the molecular details of these processes. What are the signals that recognize that the membrane has been disrupted, and how do these signals act to initiate the critical first steps of healing? In particular, what components of the actin and microtubule cytoskeletal machineries are required to drive cellular processes critical for all elements of the repair process? What are the signals that lead to disassembly of these machineries and cessation of the repair process once healing is complete? The Drosophila embryo provides an excellent genetic model in which to systematically define the specific series of events of single-cell wound repair and identify the molecules required for each step (Abreu-Blanco, 2011).
Border Cells in the Drosophila ovaries are a useful genetic model for understanding the molecular events underlying epithelial cell motility. During stage 9 of egg chamber development they detach from neighboring stretched cells and migrate between the nurse cells to reach the oocyte. RNAi screening led to the identification of the dapc1 gene as being critical in this process. Clonal and live analysis showed a requirement of dapc1 in both outer border (oBC) cells and contacting stretched cells (SCs) for delamination. This mutant phenotype was rescued by dapc1 or dapc2 expression. Loss of dapc1 function was associated with an abnormal lasting accumulation of β-catenin/Armadillo and E-cadherin at the boundary between migrating border and stretched cells. Moreover, β-catenin/armadillo or E-cadherin downregulation rescued the dapc1 loss of function phenotype. Altogether these results indicate that Drosophila Apc1 is required for dynamic remodeling of β-catenin/Armadillo and E-cadherin adhesive complexes between outer border cells and stretched cells regulating proper delamination and invasion of migrating epithelial clusters (De Graeve, 2012). Cell migration is a dynamic process involving multiple cell-cell and cell-substrate interactions. It is therefore important to better characterize the molecular mechanisms underlying cell adhesion during all stages of cell invasion. BC migration represents a powerful in vivo model, as cells become motile through a multi-step process involving cluster assembly and cohesion, delamination from the follicular epithelium, and labile interactions with nurse cells throughout migration. All these processes require the dynamic remodeling of DE-cadherin and β-catenin/Arm during adhesion. Indeed, an artificial DE-cadherin-β-catenin/Arm fusion protein can act as a strong dominant negative preventing BC migration (De Graeve, 2012). Several mechanisms can regulate DE-cadherin - β-catenin/Arm interactions. In cell culture systems, Apc proteins have been shown to be able to compete with E-cadherin for β-catenin/Arm binding. As a result, β-catenin/Arm is continuously incorporated into and released from adherens junctions. Hence β-catenin/Arm exchange is strongly affected in cells containing mutations in the apc gene. Nevertheless, little is known about the molecular events involved in cell delamination and about the role of Apc in this process. In order to better understand how cell-cell and cell-substrate interactions control BC migration, an RNAi-based genetic screen was performed and dapc1 was identified as a key regulator of BC delamination (De Graeve, 2012). The results show that dApc1 regulates BC delamination through DE-cadherin - β-catenin/Arm remodeling at the interface between oBC and SC. Indeed, loss of dapc1 function in oBC and adjacent SC led to abnormal persistence of DE-cadherin and β-catenin/Arm proteins at their boundary preventing them to detach from neighboring cells. Down regulation of β-catenin/arm or DE-cadherin rescued the dapc1 mutant phenotype indicating that loss of dapc1 function indeed increases adhesion strength between oBC and adjacent SC. The results fit with a model in which dApc1 regulates BC-SC adhesion acting at two levels. First, dApc1 competes with DE-cadherin for β-catenin/Arm binding, hence regulating the interaction between DE-cadherin and β-catenin/Arm and thereby adhesion remodeling. Second, dApc1 also favors β-catenin/Arm degradation, thereby controlling the level of proteins involved in adhesion. Altogether, this allows dApc1 to regulate negatively global adhesion strength in between oBC and SC and control BC delamination (De Graeve, 2012). In the absence of dApc1, the half-life of β-catenin/Arm is sustained, its interaction with DE-cadherin is favored and as a consequence cells display at their surface a higher number of stable adhesions. The dapc1 over-adhesive phenotype can be rescued by lowering β-catenin/Arm levels, rendering delamination again possible. Surprisingly, overexpression of β-catenin/Arm in dapc1 mutant cells also generated a partial rescue of the dapc1 mutant phenotype. Although most of the BC clusters did not reach the oocyte in time, some mutant clusters were able to delaminate. Overexpression of β-catenin/Arm probably bypasses the need of dApc. Indeed excess of β-catenin/Arm molecules generates inter-molecular competition for DE-cadherin binding, rescuing partially BC delamination. In contrast to wild type β-catenin/Arm, the overexpression of ArmS10 did not rescue the dapc mutant phenotype. ArmS10 lacks a sequence (aa 34-87) that contains a consensus GSK-3β phosphorylation site leading to the degradation of wild type β-catenin/Arm protein after phosphorylation. This suggests that β-catenin/Arm phosphorylation by GSK3-β kinase is required for BC delamination. The results are consistent with previous data in mammals showing the presence of Apc and β-catenin/Arm containing complexes that are phosphorylated by GSK3β/CKI, favoring their degradation (De Graeve, 2012). The dapc mutant phenotype requires loss of dapc both in oBC and adjacent SC. Indeed, when mutant BCs interact with wild type SCs, or vice-versa, abnormal accumulation of β-catenin/Arm and DE-cadherin is no longer detectable in between the mutant and wild type cells and BC delaminate, migrate and reach the oocyte normally. This suggests that in the absence of dapc, DE-cadherin from the mutant cell can still establish a functional interaction with DE-cadherin from the wild type cell. The remodeling of β-catenin/Arm-DE-cadherin in the wild type cell is probably sufficient to allow the release of the mutant cell (De Graeve, 2012). Live imaging of migrating BC clusters revealed that oBC change their position within the cluster throughout migration. Loss of dapc function led to persistent β-catenin/Arm and DE-cadherin at cell boundaries (oBC-oBC and oBC-SC interfaces), therefore potentially increasing cluster stiffness. However, no defect was observed in mutant oBC tumbling or cluster velocity, suggesting that dApc1 is not essential for regulating inter-oBCs interactions during migration (De Graeve, 2012). Several models from cell culture to mouse have been used to study Apc function. Wild type Apc acts as a scaffold for many proteins including F-actin, microtubules, β-catenin/Arm, and regulates multiple biological processes independent of Wg signaling, such as chromosomal segregation, cell adhesion, cell migration and apical cell extrusion. The current results show that BC migration provides a new powerful model, out of Wg influence, unraveling mechanisms regulating collective cell migration in vivo with important implications for wound healing and tumor metastasis (De Graeve, 2012). The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. This study shows that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions (Srinivasan, 2012).
This work identifies a role for the transmembrane receptor tyrosine
phosphatase Lar, acting cell-autonomously to maintain attachment
of Drosophila male GSCs to the hub. Lar function appears to
promote the maintenance of robust adherens junctions between
GSCs and hub cells and to localize and/or retain E-cadherin at the
hub-GSC interface. Consistent with the recently demonstrated
requirement for E-cadherin to polarize GSCs by localizing Apc2 at
the hub-GSC interface and to establish centrosome orientation in
GSCs (Inaba, 2010), Apc2 was often mislocalized around the GSC cortex and centrosomes were often misoriented in Lar mutant GSCs (Srinivasan, 2012).
Lar may function in parallel with other cell signaling pathways
that are important for maintaining attachment of GSCs to the hub.
Activation of the JAK-STAT pathway in Drosophila male GSCs
maintains GSCs at the hub. However, Stat92E protein levels appeared normal in Lar mutant
GSCs, suggesting that Lar function is not required for activation of
the JAK-STAT pathway. The Rap1 GTPase/Rap1 guanine
nucleotide exchange factor (Rap-GEF) signaling pathway also
regulates hub-GSC adhesion. Like Lar mutants,
Rap-GEF mutants have impaired adherens junctions at the hub-
GSC interface resulting in GSC loss. However, Rap-GEF function
is required in hub cells, whereas Lar functions in GSCs to promote
hub-GSC adhesion. Interestingly, expression of E-cadherin-GFP in
either hub cells or GSCs of Rap-GEF mutants resulted in wild-type
numbers of GSCs and restored E-cadherin localization at the hub-
GSC interface, whereas expression of Ecadherin-GFP in Lar mutant GSCs did not rescue the loss of GSCs, suggesting that the Rap-GEF and Lar signaling pathways might use different mechanisms to build and/or maintain adherens junctions between the hub cells and GSCs (Srinivasan, 2012).
The ability of some GSCs to persist next to the hub in Lar
mutant testes might be due to partial redundancy between Lar and
other tyrosine phosphatases such as the type IIA family receptor
tyrosine phosphatase Ptp69D, which has overlapping functions
with Drosophila Lar in the central nervous system and the visual
cortex and shares common signaling mechanisms. Alternatively, weak hub-GSC
adhesion in Lar mutant testes might enable CySCs to compete for
attachment to the hub, displacing some, but not all, GSCs from the
hub. CySCs normally have smaller regions of contact with the hub
than do GSCs but can outcompete GSCs from the hub when provided with an advantage. For example, overexpression of components of the integrin-based adhesion system in CySCs resulted in displacement of GSCs from the hub by CySCs (Srinivasan, 2012).
In wild-type testes, Lar localizes to the hub-GSC interface,
which is the region of cell cortex where localized adherens
junctions anchor GSCs to their niche. Adherens junctions are formed by extended clustering of
transmembrane cadherin proteins that form homotypic interactions
with cadherins on opposing cell membranes. The highly conserved cytoplasmic tail of E-cadherin acts as an anchor for β-catenin and p120-catenin and indirectly for α-
catenin through its interaction with β-catenin. Lar also localizes to adherens junctions in epithelial cells and in neuronal synapses that are enriched in
cadherin-catenin complexes. Lar physically
associates with the cadherin-catenin complex in cultured cells and
with N-cadherin in Drosophila embryos (Srinivasan, 2012).
Adherens junctions are associated with underlying arrays of
cortical F-actin, organized by the high local
concentration of β-catenin dimers. F-actin filaments, in turn, regulate the stability and strength of adherens junctions. Biochemical and genetic analyses of Lar indicate a role in regulating the actin cytoskeleton. Loss of Lar function in
Drosophila oocytes results in defects in follicle formation, egg
elongation and anterior-posterior polarity that are correlated with
defects in actin filament organization. Lar might help to maintain hub-
GSC adhesion by interacting with and modulating the function of
regulators of F-actin. Drosophila Lar and its homologs physically
and genetically interact with Ena, a member of the Ena/VASP
family of actin regulators. Drosophila Ena and its mammalian homologs localize to adherens
junctions and have been implicated in the formation and
strengthening of adherens junctions in several cell types. However, although Ena
localized to the hub-GSC interface, where adherens junctions are
present, its function was not absolutely required for GSC
maintenance, suggesting that other F-actin regulators in addition to
Ena function to maintain hub-GSC adhesion (Srinivasan, 2012).
Lar protein localized to the hub-GSC interface might instead, or
in addition, regulate the tyrosine phosphorylation state of
components of the adherens junctions to maintain strong adhesion
between hub cells and GSCs. Regulation of the tyrosine
phosphorylation of components of adherens junctions plays an
important role in modulating the adhesive state of cells. Tyrosine phosphorylation of E-cadherin in epithelial cells induces loss of cell-cell contacts and the endocytosis of E-cadherin. A possible role of Lar is to maintain adherens junctions by dephosphorylating
E-cadherin. Alternatively, Lar might target E-cadherin to the
membrane to build adherens junctions, as has been shown for the
mammalian homolog of Lar in cultured hippocampal neurons,
where it promotes the accumulation of cadherin-catenin complexes
at the synapse to enhance cell adhesion. Alternatively, or in addition, Lar might regulate tyrosine phosphorylation of the catenins associated with E-cadherin at the
hub-GSC interface. Tyrosine phosphorylation of β-catenin leads to
loss of cadherin-β-catenin interaction and to internalization of Ecadherin,
reducing the strength of adherens junctions. Mammalian Lar has been shown to
dephosphorylate β-catenin in vitro, suggesting that in vivo Lar might
promote cell adhesion by regulating the phosphorylation of β-catenin (Srinivasan, 2012).
In addition to GSCs, Lar protein was also detected in two-, four- and
eight-cell transit-amplifying spermatogonial cysts, which have
the ability to dedifferentiate and reoccupy the hub to replace lost
GSCs. Under conditions that promote dedifferentiation,
spermatogonial cells send out dynamic, actin-rich, thin protrusions,
suggesting acquisition of motility. An intriguing possibility is that Lar might facilitate the ability of dedifferentiating spermatogonial cells to reorganize their actin
cytoskeleton to recognize and build adherens junctions with the
hub, similar to the role of Lar in the nervous system, where it
promotes axonal migration, possibly by facilitating reorganization
of the actin cytoskeleton. One of the ligands
of Lar identified in the nervous system, the heparan sulfate
proteoglycan Dally-like (Dlp), is expressed by hub cells and helps
maintain GSCs in their undifferentiated state.
At Drosophila neuromuscular junctions, Dlp interacts with and
inhibits the phosphatase activity of Lar to regulate active zone
morphology and function at synapses.
Similarly, in dedifferentiating spermatogonial cells, interaction of
Lar in GSCs with Dlp on hub cells might inhibit cell motility and
promote the formation of adherens junctions between hub cells and
the dedifferentiating germ cells (Srinivasan, 2012).
shotgun:
Biological Overview
| Evolutionary Homologs
| Regulation
| Effects of Mutation
| References
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