PDGF- and VEGF-receptor related
Similar to the Drosophila Egfr and to the mammalian PDGFR family, stimulation of PDGF- and VEGF-receptor related (Pvr) activates the MAP-kinase pathway in Schneider cells as well as in border cells. However, it has been shown, by loss-of-function and gain-of-function experiments, that MAP-kinase signaling does not affect border cell migration. In addition, no effect of phospholipase C-gamma (PLC-gamma) or phosphatidylinositol 3' kinase (PI3K) has been demonstrated on this migration, using loss-of-function mutants (PLC-gamma) or border cell expression of dominant negative and dominant activated forms (PI3K). This was somewhat unexpected, since PLC-gamma and PI3K have been implicated in motility and guidance effects of RTKs (in particular PDGFR) in tissue culture cells. To address how Pvr signaling might be affecting cell migration in vivo, the effect of Pvr signaling on cell morphology and cytoskeleton was tested. In border cells as well as in other follicle cells, expression of lambda-Pvr has a dramatic effect on the actin cytoskeleton. Massive F-actin accumulation, actin-rich extensions, and changes in cell shape were produced in lambda-Pvr expressing follicle cells. The normal cells have modest cortical F-actin accumulation. This result was likely to be relevant to the guidance function of Pvr, because direct control of F-actin accumulation would allow receptor activation to control cell migration (Duchek, 2001b).
The actin cytoskeleton has been shown to be affected by small GTPases of the Rho superfamily in many systems, with the exact effects depending on the cellular context. In the border cell migration system, Rac is an attractive candidate for mediating the effect of activated Pvr, since dominant negative Rac (RacN17) has been shown to inhibit border cell migration (Murphy, 1996). Epistasis experiments could not be done by quantifying border cell migration because activated Pvr and dominant negative Rac have the same effect. Instead, whether Rac is required for the effect of Pvr on the actin cytoskeleton in follicle cells was tested. Coexpression of dominant negative Rac suppresses the effect of activated Pvr on the actin cytoskeleton. In addition, follicle cells expressing activated Rac (RacV12) have dramatic accumulation of F-actin, resembling that caused by activated Pvr. Finally, if Rac were directly downstream of Pvr, one would expect activated Rac to inhibit border cell migration, as observed for the activated receptor. Although a previous study reported that activated Rac does not affect border cell migration (Murphy, 1996), this was reexamined using the slboGal4 driver and it was found that activated Rac completely blocks border cell migration. These results are consistent with a role of Rac in the guidance pathway downstream of Pvr (Duchek, 2001b).
In mammalian tissue culture cells, PDGF stimulation can cause Rac-dependent F-actin accumulation, suggesting that the effect observed in follicle cells may reflect a conserved pathway. PI3K has been implicated as a mediator of the effect of PDGFR on Rac in Swiss 3T3 cells. However, PI3K does not appear to play a key role in guidance of border cell migration as discussed above. To investigate how Pvr might lead to activation of Rac, two groups of Drosophila mutants were tested for their effect on border cell migration: mutants in genes shown to be downstream of receptor tyrosine kinases in other contexts, and mutants linked to Rac activation. Most mutations were homozygous lethal, so their effect in border cells was tested by generating mutant clones in a heterozygous animal (mosaic analysis). Of the 8 genes tested, only myoblast city (mbc) has a detectable effect on border cell migration. Mbc is homologous to mammalian DOCK180 and C. elegans CED-5. Mbc/DOCK180/CED-5 acts as an activator of Rac (Kiyokawa, 1998; Nolan, 1998; Reddien, 2000, cited in Duchek, 2001b).
mbc has been independently identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. slbo mutant border cells migrate poorly. Increased expression of mbc in slbo mutant border cells improves their migration, suggesting that mbc has a positive role in promoting border cell migration. Mbc protein is detected in follicle cells, including border cells, and is overexpressed upon induction of the EP element EPg36390 located upstream of mbc. Removing mbc function from border cells by generating mutant clones causes severe delays in their migration. At stage 10, when 100% of control (GFP) clones have reached the oocyte, only 10% of mbc mutant border cell clusters have done so, and these are the oldest egg chambers. Thus, mbc is not absolutely required for border cell migration, but, contrary to the other genes implicated in RTK and Rac signaling, loss of mbc function severely impairs this cell migration (Duchek, 2001b).
To test whether Mbc can act downstream of Pvr, focus was placed on the effect of lambda-Pvr on F-actin accumulation in follicle cells. In order to obtain mbc mutant clones in egg chambers that express lambda-Pvr (constitutively active Pvr), the experiment was done slightly differently from that for dominant negative Rac. Expression of lambda-Pvr under control of slboGal4 causes disruption of centripetal cell morphology and abnormal actin accumulation. When follicle cells are mutant for mbc, this effect is strongly attenuated, indicating that Mbc acts downstream of Pvr. Taken together, these results suggest that Pvr affects guidance of border cell migration, at least in part, by signaling through Mbc to Rac, which then controls F-actin accumulation (Duchek, 2001b).
Invasive cell migration in both normal development and metastatic cancer is regulated by various signaling pathways, transcription factors and cell-adhesion molecules. The coordination between these activities in the context of cell migration is poorly understood. During Drosophila oogenesis, a small group of cells called border cells (BCs) exit the follicular epithelium to perform a stereotypic, invasive migration. The ETS transcription factor Yan is required for border cell migration and Yan expression is spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor tyrosine kinase pathways (Egfr and Pvr) in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. These results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells (Schober, 2005).
Interestingly, Yan expression levels gradually decrease as BCs move along an increasing PVR/EGFR activity gradient. Yan has been shown to be phosphorylated by the EGFR-MAPK pathway, which triggers its nuclear export and protein degradation. Consistent with these previous studies, expression of dominant-active PVR and EGFR in BCs blocks BC migration and abrogates Yan protein expression, whereas yan transcript or enhancer trap expression is still detectable. Expression of activated Ras and Raf similarly induced Yan downregulation, consistent with an involvement of the canonical Ras/MAPK pathway in mediating PVR/EGFR signaling. It is noted, however, that although BC migration is significantly delayed upon ectopic expression of activated Ras, activated Raf hardly affects their ability to migrate. The basis of this difference, which might be due to complex feedback loops between the implicated signaling pathways, is unclear at the present time and will need to be investigated further (Schober, 2005).
Schneider cells were used to determine whether Pvf1 could bind to and activate Pvr. The anti-Pvf1 antibody detected a specific band of about 36 kDa in conditioned medium from Schneider cells. This appears to be secreted Pvf1, since a stronger signal at the same position was observed in conditioned medium from Schneider cells transfected with an expression construct for Pvf1 (pRm-Pvf1). For binding studies, a Pvf1-Alkaline Phosphatase (AP) fusion protein was produced in Schneider cells and harvested in conditioned medium. The Pvf1-AP fusion protein binds to Schneider cells in a Pvr-dependent manner, since binding is significantly decreased by pretreatment of the cells with Pvr dsRNA. The remaining binding of Pvf1-AP to cells may be nonspecific sticking or binding to other proteins. To look at consequences of Pvf1 binding to Pvr, MAP-kinase activation was monitored in recipient cells by anti-diphospho-ERK (anti-dpERK) staining. MAP-kinase is activated by conditioned medium containing Pvf1. This activation is abolished by prior treatment of the cells with Pvr dsRNA. Maximal activation was observed in 10 min. Thus, Pvf1 binds to Pvr, and Pvr activates the MAP-kinase pathway in Schneider cells (Duchek, 2001b).
A vascular endothelial growth factor (VEGF) pathway controls embryonic migrations of blood cells (hemocytes) in Drosophila. The VEGF receptor homolog is expressed in hemocytes, and three VEGF homologs are expressed along hemocyte migration routes. A receptor mutation arrests progression of blood cell movement. Mutations in Vegf17E (CG7103; also referred to as Pvf1) or Vegf27Cb (CG13780; also referred to as Pvf2) have no effect, but simultaneous inactivation of all three Vegf genes, including Vegf27Ca (CG13781/13782; also referred to as Pvf3) phenocopies the receptor mutant, and ectopic expression of Vegf27Cb redirects migration. Genetic experiments indicate that the VEGF pathway functions independently of pathways governing hemocyte homing on apoptotic cells. The results suggest that the Drosophila VEGF pathway guides developmental migrations of blood cells, and it is speculated that the ancestral function of VEGF pathways was to guide blood cell movement (Cho, 2002).
Blood cells (hemocytes) in Drosophila migrate extensively during development. They originate in the head mesoderm, and over a 7 hr period in midembryogenesis they migrate along specific pathways to disperse throughout the body, where they function as immune and interstitial cells. Like vertebrate monocytes and macrophages, insect hemocytes phagocytose or encapsulate foreign material and apoptotic cells. This is important during development because cell death is widespread, and as hemocytes disperse through the embryo they recognize and remove cell remnants. Hemocytes also produce many extracellular matrix molecules, including collagen IV and laminin, that compose the basement membrane surrounding internal organs. Although there has been progress in understanding the genetic control of blood cell differentiation in Drosophila and the ability of blood cells to recognize and engulf dying cells, little is known of the genetic and molecular mechanisms controlling their developmental migrations. It is also unclear how developmental migrations are coordinated with hemocyte homing toward dying cells along the migration pathway (Cho, 2002).
A large-scale mutagenesis was carried out by mobilizing a piggyBac[w+] transposable element. Inverse PCR and DNA sequencing of piggyBac[w+] insertion sites have identified three lines with insertions in Vegfr. Vegfrc2195 is a homozygous lethal insertion located within the small (67 base) 11th intron. RNA in situ hybridization has demonstrated that Vegfr transcript is undetectable in Vegfrc2195 embryos, and genetic studies indicate it is an amorphic allele (Cho, 2002).
Vegfrc2195 mutants display a striking defect in hemocyte migration. Formation of hemocytes and their initial migrations are normal, as judged by staining of Croquemort (CRQ) and Peroxidasin (PXN). By stage 11, posteriorly directed hemocytes reach the caudal margin normally. However, unlike Vegfr+ blood cells, which rapidly enter the tail, blood cells in mutant embryos never enter the region, instead accumulating at the caudal margin. By stage 13, wild-type hemocytes are dispersed throughout the embryo, whereas mutant hemocytes have clumped together in aggregates concentrated in the anterior. The mutant blood cells continue to express CRQ and PXN, suggesting that hemocyte differentiation is grossly intact. Interestingly, CRQ staining is stronger than in wild-type, even in isolated blood cells, implying their phagocytic function is activated. Aside from the hemocyte defects, the mutant embryos appear normal, and no defects are detected in the CNS, muscles, and tracheal system after staining with tissue-specific markers (Cho, 2002).
The severity of the hemocyte phenotype of Vegfrc2195 homozygotes is the same as that of Vegfrc2195 hemizygotes and homozygous deficiency embryos, implying that Vegfrc2195 is an amorphic allele. Vegfrc2859, a lethal piggyBac[w+] insertion in the first intron, and Vegfrc3211, a homozygous viable insertion in the 3' noncoding region, do not exhibit the hemocyte phenotype. However, the disruption of hemocyte migration clearly reflects a requirement for Vegfr function, since the migration defect is also seen when endogenous Vegfr transcripts are depleted by RNAi. It is concluded that inactivation of the Vegfr gene blocks progression of blood cell movement; hence, the gene name stasis (stai, pronounced 'stay'), which means slowing or stopping, is proposed (Cho, 2002).
The RAS-MAPK pathway is activated by signaling through VEGFRs and other RTKs, so whether RAS-MAPK is involved in hemocyte migration was investigated. Immunostaining with a diphospho-MAPK antiserum shows that MAPK is activated in migrating hemocytes. MAPK activation is greatly reduced in Vegfr mutants, and it is increased by ectopic expression of a VEGF ligand. It was not possible to test the effect of complete loss of RAS-MAPK pathway activity in embryonic hemocytes, because pathway components are both maternally and zygotically required and expressed. Zygotic loss of function mutations in any of the three genes encoding adaptor proteins (drk, dshc) or a RAS exchange factor (sos) have no effect on hemocyte migration. However, expression of a dominant-negative RAS protein (DRAS1N17) in hemocytes causes an early migration arrest similar to that seen in the Vegfr mutant, implicating RAS in the process (Cho, 2002).
BLAST searches have identified three genes encoding proteins with sequence similarity to vertebrate VEGFs. Vegf27Ca (CG13781/13782; also referred to as Pvf3) and Vegf27Cb (CG13780; also referred to as Pvf2) are adjacent genes at cytological position 27C. The genes are tandemly arrayed, separated by ~16 kb. Their close proximity, sequence similarity, and nearly identical expression patterns suggest they have been generated by a recent gene duplication. The VEGF homolog at cytological position 17E (Vegf17E; CG7103; also referred to as Pvf1) produces two splice variants (A and B) that differ by 11 N-terminal residues (Cho, 2002).
All the Drosophila VEGF proteins have a predicted signal peptide and central domain common to VEGF/PDGF superfamily members. All also have a cysteine-rich C-terminal domain, as do vertebrate VEGF-C and VEGF-D, but lack the C-terminal heparin binding domain found in human VEGF-A and VEGF-B. VEGF17E is slightly more similar to vertebrate VEGFs than to PDGF, whereas VEGF27Ca and VEGF27Cb are equally similar to both (Cho, 2002).
Embryonic expression patterns of Vegf genes were analyzed by RNA in situ hybridization. The genes are expressed in dynamic spatial and temporal patterns that line many of the migratory paths of developing blood cells. Vegf17E begins to be expressed at the end of stage 10 in an ectodermal patch at the caudal margin of the germband where blood cells enter the tail. It is also expressed in the developing trachea and salivary glands. General tracheal expression persists through stage 12, after which it restricts to the tips of growing ganglionic branches and more strongly in the visceral branches. The latter is the site where a novel population of Vegfr-positive cells cluster in the embryo. From stage 12 on, Vegf17E is expressed in Malpighian tubules, and beginning at stage 13, in a posterior ring of ectodermal cells (Cho, 2002).
Vegf27Ca and Vegf27Cb are also expressed along blood cell migration routes. Both display the same expression pattern. Beginning at stage 9 and into stage 11, the genes are expressed in caudal ectoderm and developing hindgut, foregut, and ventral nerve cord. The hindgut (and subsequent Malpighian tubule) expression during stages 11-13 corresponds precisely to the position where blood cells cluster after entering the tail. There is also a striking correlation between the location of Vegf27Ca/27Cb expression and Vegfr-expressing blood cells at the ventral nerve cord, along which hemocytes move to reach the middle of the embryo. In late embryogenesis, Vegf27Ca/27Cb expression is detected in Malpighian tubules and a foregut derivative, both of which are also associated with migrating hemocytes (Cho, 2002).
In aggregate, the expression pattern of the three Vegf genes coincides with many blood cell migratory paths. This correlation is especially clear at the entry site into the tail and along the ventral midline, paths that Vegfr- hemocytes fail to engage (Cho, 2002).
To determine if Vegf genes are required for hemocyte migration, Vegf17E and Vegf27Cb mutants were isolated. Three Vegf17E mutants were examined, including Vegf17Eex3.6, a transcript null allele. No blood cell migration defects were detected in any of the mutants. A piggyBac[w+] insertion in Vegf27Cb, Vegf27Cbc6947 was characterized. It is a homozygous viable insertion five nucleotides downstream of the 5' splice site of the fourth intron. This would likely disrupt exon 4-5 splicing and prevent inclusion of C-terminal coding sequences. No hemocyte migration defects were detected (Cho, 2002).
The above results suggested that if Vegf ligands are required for blood cell migration, they are likely to be redundant. To test for redundancy, RNAi was used to inactivate multiple Vegf genes simultaneously. As a control, it was first demonstrated that inactivation of Vegfr by RNAi could phenocopy the blood cell migration defects in Vegfr mutants. 59% of embryos showed mild (class I) to severe (class III) defects in hemocyte migration, with 24% of affected embryos showing a severe phenotype similar to that observed in null Vegfr mutants. RNAi of either Vegf27Ca or Vegf27Cb alone has little effect above background, and simultaneous RNAi of both Vegf27Ca and Vegf27Cb has only a moderate effect. Simultaneous inactivation of all three Vegf genes, however, results in a defect very similar to Vegfr inactivation: 71% of injected animals showed blood cell migration defects, with 14% of affected embryos showing the extreme phenotype. It is concluded that Vegf ligands are redundantly required for blood cell migration, and they are required for the same function as Vegfr (Cho, 2002).
The Gal4/UAS system was used to test if misexpression of a Vegf ligand could alter hemocyte migration. Misexpression of Vegf27Cb in the developing foregut, salivary duct, trachea, and midline glia using breathless-Gal4 driver (btl-Gal4) and UAS-Vegf27Cb (XP d2444) causes misrouting of hemocytes. In many embryos, most blood cells are redirected to anteroventral positions on and around the foregut, the site of ectopic expression closest to where they originate. Similar experiments using btl-Gal4 and XP d5686 to misexpress Vegf17E did not show an effect on hemocyte migration, nor did experiments using a UAS-Vegf17E transgenic line (UAS-Vegf17E-B). This suggests that the activity or diffusion properties of VEGF17E ligands may differ from those of VEGF27Cb (Cho, 2002).
The only gene previously known to alter the developmental migration of hemocytes is singleminded, a transcription factor that controls ventral midline development. In sim- embryos, ventral midline cells do not develop normally, and hemocytes do not migrate along this tissue. Ventral midline expression of Vegf27Cb is selectively eliminated in sim- embryos. Thus, sim functions upstream of Vegf27Cb in control of blood cell migration in the nervous system (Cho, 2002).
The simplest model of how VEGF signaling controls blood cell migration is that VEGFs serve as chemoattractants for blood cells expressing VEGFR. Vertebrate VEGFs function in vitro as chemoattractants for leukocytes and blood vessel enthothelial cells, and the ligand-expressing cells in Drosophila are located at the entry site to the tail and along most hemocyte migratory routes in the posterior. Thus, they are perfectly positioned to guide hemocytes along these routes. Not only are Vegf ligand genes required for migration, but ectopic expression of one of them (Vegf27Cb) in the foregut caused rerouting of blood cells to this tissue, demonstrating that localized expression of the ligand provides guidance information. Duchek (2001) proposes a similar role for VEGF17E in border cell migration in the egg (Cho, 2002).
In the chemoattraction model, VEGFs guide most blood cell migrations into and around the posterior. Because there are multiple sites of Vegf gene expression, the question arises as to how cells progress from one VEGF source to another. What causes them to leave the first source encountered? Perhaps ligand expression is highly dynamic, turning off transiently after a blood cell arrives, or perhaps a cell's arrival triggers mechanisms that selectively desensitize the cell to ligand produced from that source. The different ligand and receptor isoforms could also play a role if they have different functional properties, as suggested by misexpression studies with Vegf27Cb and Vegf17E. There also could be auxiliary factors that promote blood cell movement away from one VEGF source and on to the next (Cho, 2002).
VEGF pathway mutants divide blood cell migration into three phases. The Vegf expression patterns and loss of function phenotype suggest that VEGF signaling controls many instances of migration of blood cells, particularly those in and around the posterior. But the results also imply involvement of other signaling pathways before and after their arrival at the posterior. In Vegfr-embryos, the initial migration of hemocytes to the caudal margin is unaffected. Anteriorly and ventrally directed migrations during these stages also appear grossly normal. This defines an early, Vegf-independent phase of migration (Phase I). Also, the late dispersal of hemocytes is not associated with Vegf expression, defining another Vegf-independent phase (Phase III). It will be important to identify the pathways that control these early and late migrations and learn how they are coordinated with the VEGF pathway. It will also be of interest to explore the function of VEGF signaling in the few domains of Vegf expression not obviously associated with blood cell migration (Cho, 2002).
Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-ß signalling. Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp. Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal (Jékely, 2003).
Monoubiquitination of membrane proteins has an important role in regulating their internalization and sorting to lysosomal degradation. The ubiquitin tag is recognized by proteins containing a ubiquitin interaction motif (UIM), such as epsins, Hse1p/STAM and Eps15. Hrs and its budding yeast homolog, Vps27p, also have a UIM and bind to ubiquitin. The ubiquitin-binding ability of Hrs and Vps27p is required for the efficient sorting of ubiquitinated transferrin receptors in mammalian cells and Fth1p in yeast (Jékely, 2003 and references therein).
To determine whether Hrs is generally required for sorting and degradation of ubiquitinated proteins in Drosophila tissues, clones of cells mutant for hrs were generated within an epithelium using somatic recombination. Follicle cells of the Drosophila ovary and wing imaginal disc cells from third instar larvae were examined. Follicular cells form a simple monolayer epithelium surrounding the germline cells and are large enough to detect subcellular localization of protein. The imaginal disc cells are smaller and form a pseudo-stratified epithelium. The mosaic tissues were stained with an antibody that recognizes mono- and poly-ubiquitinated proteins. Both follicle cells and wing disc cells lacking Hrs show a dramatic accumulation of ubiquitinated proteins. Most of the signal localizes to intracellular structures. In some cases accumulation at the cell cortex could also be observed. Thus, Hrs is required for the efficient removal of ubiquitinated proteins from the cell (Jékely, 2003).
An enlarged vesicular structure, the 'class E' compartment, has been observed in yeast cells mutant for VPS27. Genetic studies in mice and Drosophila (Lloyd, 2002) have also shown that cells mutant for hrs have enlarged endosomes, possibly due to impaired membrane invagination and multivesicular body (MVB) formation (Lloyd, 2002). To determine whether ubiquitinated proteins accumulate in the endosomal compartment in hrs mutant cells, GFP-Rab5 or GFP-2xFYVE fusion proteins were expressed in hrs mutant cells. Rab5, a small GTPase regulating endosome fusion, is a marker of early endosomes. FYVE domains bind to phosphatidylinositol-3-phosphate, which is enriched in endosomal membranes, and can also be used to specifically label endosomes. The ubiquitinated protein signal and the GFP-2xFYVE signal show extensive overlap in hrs mutant follicle cells. GFP-Rab5 and ubiquitinated proteins also show significant, although not complete, overlap. These data indicate that nondegraded ubiquitinated proteins accumulate in the endosomal compartment. Additionally, when the GFP-2xFYVE signal in hrs mutant and nonmutant cells is compared, an enlargement of FYVE-positive structures is observed in mutant cells, consistent with an enlargment of the endosomal compartment (Jékely, 2003).
Hrs affect degradation of receptor tyrosine kinases (RTKs). Indeed the two RTKs that were analysed in follicle cells, EGFR and PVR (PDGF/VEGF receptor), accumulate within hrs mutant cells, mostly in intracellular structures. These structures were also positive for the ubiquitinated protein signal, indicating that the receptors accumulate in endosomes (Jékely, 2003).
Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. Complete loss-of-function mutants of the single Drosophila Cortactin gene have been isolated. Mutants are viable and fertile, showing that Cortactin is not an essential gene. However, Cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In Cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). Accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. Evidence is presented that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the Cortactin gene not being essential for development (Somogyi, 2004).
A number of specific defects were observed in cortactin mutants. One cortactin phenotype is a mild defect in 'dumping', transfer of bulk cytoplasmic material from nurse cells to the oocyte. This phenotype is similar to that observed in Src64 mutants, but is also seen in other mutants. In the case of Src64, the defect is correlated to the presence of Src64 protein at the ring canals and to reduced size of the ring canals. Cortactin is also present at ring canals. To determine whether cortactin affects ring canal morphogenesis, their size was quantified at stage 10. Ring canal size is significantly reduced in the cortactin mutant relative to wild type. Dumping defects usually result in smaller eggs. Eggs from cortactin mutant mothers and from Src64 mutant mothers are on average smaller than wild type. The hatching rate of the eggs from mutant mothers is also decreased. Thus, mutations in cortactin affect the actin rich ring canals, and processes dependent on function of ring canals, in a manner similar to mutations in Src64. Mutations in components of the Arp2/3 complex (Arpc1 or Arp3 mutants) are also known to specifically affect ring canal morphogenesis (Somogyi, 2004).
There were several reasons to suspect that the effect of Cortactin on the actin cytoskeleton could be related to effects of RTKs. Border cell migration is guided by two RTKs, namely PDGF/VEGF receptor (PVR) and EGFR. Also, an activated form of PVR induces robust formation of actin-rich extensions in follicle cells in a Rac dependent manner. Finally, mammalian Cortactin has been suggested to act as a link between RTKs such as PDGF receptor and the actin cytoskeleton. Therefore the effect of PVR signaling on Cortactin protein was examined in the follicular epithelium. Overexpression of wild type PVR is sufficient to increase signaling in follicle cells slightly, resulting in a small increase in F-actin accumulation in the cell. This is most visible at the basal F-actin network. PVR overexpression also results in clear recruitment and/or stabilization of Cortactin at the cell cortex. Cortactin protein is not simply recruited by the increased amount of F-actin; the subcellular localization of Cortactin is distinct from that of F-actin. In addition, the level and the localization of other actin-associated proteins such as moesin and alpha-spectrin are not visibly affected by PVR overexpression. Expression of a constitutive active form of PVR (lambda-PVR) results in more robust F-actin accumulation but also disruption of the normal cell shape. The activated receptor is not restricted to the cell cortex but is present in vesicles throughout the cell. The constitutive active PVR also induces accumulation of Cortactin throughout the cell. Thus, PVR activation in follicle cells affects the accumulation and subcellular localization of Cortactin protein, primarily resulting in more Cortactin at the cell cortex of normal epithelial cells (Somogyi, 2004).
The ability of Cortactin to induce F-actin accumulation and filopodia formation in conjunction with the effect of PVR on Cortactin protein suggested that Cortactin might act downstream of PVR with respect to control of the actin cytoskeleton. To determine if this might be the case, an epistasis experiment was performred. The effect of activated PVR (lambda-PVR) on F-actin accumulation and cell shape in follicle cells was scored using three categories of severity. Quantification was done by blindly scoring severity of the phenotype in many egg chambers. In each experiment follicle cells that were mutant for cortactin were compared to a control, wild type background. A small, but statistically significant decrease was seen in the severity of the lambda-PVR induced phenotypes in the cortactin mutant background. This result is consistent with Cortactin acting downstream of PVR, but also shows that the effect of PVR on the actin cytoskeleton does not strictly require Cortactin. Another factor that appears to act downstream of PVR is the Rac activator Mbc (related to DOCK180 and Ced-5). In the same assay, removal of mbc has a much more pronounced effect. Activation of Rac can cause translocation of Cortactin to the cell periphery in mammalian cells. Thus, cortical Cortactin accumulation could be one of the downstream effects of Rac activation by PVR. In conclusion, Cortactin appears to contribute to the effects of PVR on actin, but is largely redundant with other factors. A contributing, but not essential, function is also consistent with cortactin not being an essential gene (Somogyi, 2004).
Expression of an activated form of Src (Src42CA) in border cells and other follicle cells shows a phenotype similar to that of activated PVR. It completely blocks border cell migration and disrupts cell shape and the actin cytoskeleton of follicle cells. Src64 has a specific function in the female germ line, which coincides with a function of Cortactin as discussed above. Src42 appears to function more generally in somatic cells and is required for viability. Unfortunately, it is technically not feasible to make Src42 mutant clones to determine whether Src42 function is required in border cells. However, whether Cortactin might act downstream of activated Src42 could be examined by performing an epistasis experiment equivalent to that with activated PVR. Removal of cortactin results in a small, but significant, decrease in the severity of the activated Src-induced phenotype. This is consistent with a role of Cortactin downstream of Src. It also shows that Src can affect the cytoskeleton independently of Cortactin (Somogyi, 2004).
It is perhaps surprising that Cortactin in not an essential gene. Many modulators of essential processes in the cell such as dynamics of the cytoskeleton, cell adhesion or cell signaling are very well conserved in higher eukaryotes. They may add to the robustness and fidelity of the regulation, but they may only be essential to the organism if their absence completely changes the behavior or fate of specific, important cells. In mammals, there are often multiple closely related genes and simple redundancy between these gene products may explain an absence of phenotypes in knockout mice. In Drosophila, this type of simple redundancy is less frequent. For example, there is no evidence for another Cortactin gene in the sequenced Drosophila genome. However, more distantly related genes may have overlapping functions. Subtle phenotypes may also reflect that one process can be regulated in multiple ways. Combining multiple mutations can then be used to genetically help define which genes and pathways overlap in function (Somogyi, 2004).
Guidance receptors detect extracellular cues and instruct migrating cells how to
orient in space. Border cells perform a directional invasive migration during
Drosophila oogenesis and use two receptor tyrosine kinases (RTKs), EGFR and PVR
(PDGF/VEGF Receptor), to read guidance cues. Spatial localization
of RTK signaling within these migrating cells is actively controlled. Border
cells lacking Cbl, an RTK-associated E3 ubiquitin ligase, have delocalized
guidance signaling, resulting in severe migration defects. Absence of Sprint, a
receptor-recruited, Ras-activated Rab5 guanine exchange factor, gives related
defects. In contrast, increasing the level of RTK signaling by receptor
overexpression or removing Hrs, an endosome-associated, ubiquitin binding protein required for multivesicular body formation and degradation of RTKs,
and thereby decreasing RTK degradation, does not
perturb migration. Cbl and Sprint both regulate early steps of RTK endocytosis.
Thus, a physiological role of RTK endocytosis is to ensure localized
intracellular response to guidance cues by stimulating spatial restriction of
signaling (Jekely, 2005).
It was reasoned that regulation of RTK turnover might be important to maintain a
directional response in border cells, and the effects of mutations
likely to affect this process were analyzed. Cbl is a ubiquitin ligase with a conserved role in regulating RTK signaling. Clones of
border cells mutant for Cbl are correctly specified, as judged by
staining for Slbo, a marker specific for differentiated border cells, but have
severe migration defects. To explore the relationship between Cbl and
RTK signaling, RTK signaling was manipulated in Cbl mutant border cells.
The migration defect in Cbl mutant cells was suppressed by reducing the
level of an EGFR ligand (grk/+) and was enhanced by overexpression of
either receptor in border cells (UAS-PVR or UAS-EGFR.
Note that overexpression of either PVR or EGFR alone has no effect.
This indicates that the Cbl phenotype is
not due to lack of guidance signaling but instead due to excessive, misregulated
RTK signaling (Jekely, 2005).
Consistent with the
interpretation that Cbl is required to restrict signaling, the complete failure
of many Cbl mutant border cell clusters to migrate resembles the effect
of increased guidance receptor signaling due to expression of constitutively
active receptors or a strong ligand. In contrast, border cells lacking Pvr and Egfr all
eventually initiate migration but never make it to the oocyte.
Similar phenotypic effects of manipulating guidance
cues have been observed by live imaging of germ cell migration in the zebrafish
embryo, a migration guided by a G protein-coupled receptor: migratory
cells lacking guidance cues migrate, but randomly, whereas the same cells
subject to high uniform guidance cues did not migrate. Thus, border cells
lacking Cbl responded as if they were receiving high uniform signaling, a
situation that mimics the endpoint of migration (Jekely, 2005).
Mammalian Cbl proteins
negatively regulate multiple RTKs by stimulating their ubiquitination and
lysosomal degradation. The N-terminal
phospho-tyrosine binding domain of Cbl directly binds to activated receptors,
and ubiquitin-conjugating enzymes are recruited via the E3 type RING finger.
The N-terminal part of Drosophila Cbl also physically
interacts with autophosphorylated Pvr. In some assays, these conserved domains
are sufficient for mammalian Cbl to regulate Egfr. Cbl can also interact with proteins
regulating endocytosis as well as other signaling molecules through its less
well conserved C-terminal region. Drosophila Cbl is expressed
as two isoforms. Ubiquitous expression of
either Cbl-L or Cbl-S, which lacks the C-terminal tail, rescues lethality
associated with the Cbl null mutation as well as the migration phenotype.
To determine whether E3 ligase activity of Cbl
is required, a single cysteine residue essential for this activity
was mutated to alanine
(Cys-369, corresponds to Cys-381 in human Cbl). The Cbl ring finger mutant was
unable to rescue viability of the Cbl mutant or migration of Cbl
mutant border cell clones, showing that this
function is essential for Cbl activity during migration (Jekely, 2005).
Overall level
of RTK activity, in the cell or at the cell cortex, does not need to be precisely
controlled to allow migration. Yet Cbl is apparently required to restrict RTK
activity. This prompted examination of whether Cbl might affect subcellular
localization of RTK signaling at a more refined level. Experiments with
Hrs mutants and RTK overexpression suggest that total phospho-tyrosine
might be used as a local indicator of RTK signaling. Many proteins are
tyrosine-phosphorylated in cells by a number of kinases, but the RTKs have a
quantitatively significant effect (direct and indirect). Remarkably, wild-type
border cells initiating migration show a clear localization of
phospho-tyrosine signal to the front.
The front is the side facing the direction of subsequent migration and
the source of the ligands, namely the oocyte. Since border cells migrate as a tight
cluster of cells, several cells contribute to the front. To further test whether
the signal reflected stimulation of endogenous RTKs, a strong Egfr ligand
(secreted Spitz) was expressed in border cells to stimulate the endogenous
receptor uniformly. This resulted in delocalized phospho-tyrosine signal all
over the cortex of the border cells,
and, as expected, a block in directed migration (70% nonmigrating clusters).
This validated the use of phospho-tyrosine as a reasonable local readout of
endogenous RTK activation (Jekely, 2005).
Endogenous Pvr and Egfr are detected at low uniform levels in border cells.
Overexpression of Egfr or Pvr
results in high level of receptor throughout the cluster; however, the
phospho-tyrosine signal remains localized.
Thus, local activation is maintained despite RTK overexpression.
Consistent with signal location being the critical parameter for guidance
signaling, directed migration also proceeds normally upon RTK overexpression.
In contrast, border cells mutant for Cbl
show a high frequency of delocalized phospho-tyrosine signal.
These results indicate that Cbl is important in migrating cells because
it is required to restrict RTK signaling spatially within the cell; without Cbl,
signaling becomes delocalized. Since Cbl affects RTK endocytosis, whether
perturbing endocytosis more generally would have the same effect was tested.
Expression of
a dominant-negative form of Shibire (dynamin) in border cells initiating
migration also caused efficient delocalization of the phospho-tyrosine signal (Jekely, 2005).
The incomplete penetrance of the Cbl phenotype suggests that other molecules might partially compensate for the loss of Cbl. Indirect evidence is available that another potential RTK binding endocytosis regulator called Sprint might have a role in border cells. The mammalian counterpart of Sprint, called RIN1, displays Ras-activated Rab5 guanine nucleotide exchange factor (GEF) activity and can bind Egfr and stimulate Egfr activity. RIN1 also binds and
activates the Abelson tyrosine kinase. To analyze the function of Drosophila
sprint in vivo, sprint mutants, including a complete
loss-of-function mutant, were generated. Despite
sprint being the only rin1-related gene in Drosophila,
homozygous sprint mutant flies are completely viable and fertile with
normal oogenesis. To determine whether Sprint might contribute to regulating
RTKs during border cell migration, the cells were challenged by overexpressing Pvr
or Egfr in the mutant background. By itself, this overexpression has no effect
on migration. In the sprint mutant
background, however, RTK overexpression results in significant migration
defects and, as for Cbl mutants, a
corresponding increase in delocalized phospho-tyrosine signal. This suggests that Sprint might play a role
similar to Cbl. Sprint might not be essential under normal conditions due to
overlap in function with Cbl. To test this further, border cells
mutant for both sprint and Cbl were analyzed. These cells have very severe
migration defects and rarely reach the oocyte.
For comparison, almost half the Cbl single mutant clusters reach the
oocyte. Since sprint has barely any defect
on its own, this strong enhancement of the Cbl phenotype is significant.
Such a synergistic effect of two null mutants indicates that the gene products
function in parallel to regulate the same process (Jekely, 2005).
To understand more about how Sprint might function in vivo, an antibody was generated that detects
endogenous Sprint. In a pattern strikingly
similar to the wild-type polarized phospho-tyrosine signal,
endogenous Sprint was detected at the front of
border cells initiating migration. This is
consistent with Sprint being recruited to active RTKs. This was confirmed by the
ability of overexpressed Egfr or Pvr to recruit endogenous Sprint.
In overexpression experiments, it was also found that Sprint has
the characteristics expected from its homology to RIN1:
Sprint binds Ras-GTP recruited Abelson kinase to the cell cortex and associates
with endocytic vesicles. Finally, endogenous Sprint
accumulates at the apical cortex of follicle cells, contacting the oocyte, upon
transient block of endocytosis. Since endogenous Egfr
and Pvr ligands come from the oocyte, this supports the idea that Sprint
dynamically associates with early endocytosis of RTKs at the cell cortex. Taken
together with the genetic analysis, it is concluded that Cbl and Sprint both serve
to maintain RTK signaling localized for guidance, although they stimulate early
endocytosis events in molecularly unrelated ways (Jekely, 2005).
Regulators such as Cbl and Sprint might be recruited directly
to activated and autophosphorylated RTKs or might bind indirectly, via
phosphorylated adaptor proteins. A yeast two-hybrid assay was set up to detect
possible direct, phosphorylation-dependent binding. The intracellular domain of
Pvr is able to autophosphorylate in yeast and bind SH2 and PTB domain proteins.
Binding was detected of both Cbl and Sprint to Pvr, but not a kinase-dead Pvr
mutant. Potential docking tyrosines in Pvr were systematically mutated.
Mutation of 16 or 14 (YF14) tyrosines results in
strong decrease in binding of Cbl and Sprint. To
map the binding sites, 5 tyrosines at a time were 'added back' to
the YF14 mutant. Although there was no overlap in tyrosines, two of the
resulting constructs regained full binding to Cbl and Sprint,
indicating that both proteins have more than one direct binding site on Pvr. One construct (YFc)
did not bind either Cbl or Sprint directly and therefore seemed to be a
potentially useful tool to study the role of their direct binding to Pvr in
vivo (Jekely, 2005).
To determine the
signaling potential of each Pvr mutant in vivo, the mutations were placed in the
context of a constitutively active form of Pvr (λ-Pvr) to induce full,
unregulated activation. The ability to block border cell migration and induce
F-actin accumulation was monitored. The activity of YF14 was strongly
reduced, but each add back mutant had only slightly reduced
activity relative to wild-type despite missing nine potential docking tyrosines.
Thus, each of the add back mutants was
still capable of signaling to affect migration and guidance when artificially
activated (Jekely, 2005).
To determine whether the YFc mutations affected receptor
regulation, they were placed in the context of full-length Pvr. From transgenes,
Pvr and Pvr-YFc were expressed in the ovary at similar levels.
As expected, the ability to activate signaling in
border cells (measured by anti-dpERK staining) was quantitatively reduced in
Pvr-YFc compared to Pvr. This result was
confirmed using the sensitive MAP kinase-activated reporter gene
kekkon-lacZ. However, expression of Pvr-YFc but not Pvr
caused border cell migration defects. Although
the frequency of defects was low, finding a gain-of-function activity at all was
significant, given that the signaling strength of Pvr-YFc was reduced compared
to Pvr. The migration defects were qualitatively similar to those of Cbl
mutants and distinct from dominant-negative effects, which even in their
strongest form cause migration delays but not arrest. Uniform
expression of the ligand PVF1 did not further affect the phenotype of Pvr-YFc,
indicating that this form of Pvr that cannot
bind Cbl and Sprint had already lost its spatial information. Consistent with
this, expression of Pvr-YFc also induced a delocalized phospho-tyrosine signal
at a frequency corresponding to the migration
defects. This analysis of Pvr itself further indicates that the phenotypes of
Cbl and sprint mutant border cells are due to their effects on RTK
signaling: recruitment of Cbl and Sprint to Pvr serves to regulate Pvr guidance
signaling, specifically to keep it localized (Jekely, 2005).
Thus, Cbl and
Sprint are required to keep RTK signaling properly localized. To show this,
phospho-tyrosine was used as a read-out of local RTK signaling. Although this
reagent is not uniquely specific for the active receptor, the visualized effects
of Pvr or Egfr overexpression, Hrs mutation, ligand misexpression, as
well as Sprint colocalization, validates its utility in visualizing the high
level of local receptor activity found at the leading edge of migrating border
cells. The requirement for Cbl and Sprint suggests that the cellular activity
required for signal restriction is receptor endocytosis, which is supported by
experiments with dominant-negative dynamin. Thus, in this physiological context
of guidance by RTKs, receptor endocytosis serves not to downregulate active
receptors, but to ensure their correct spatial localization (Jekely, 2005).
The proposed role
of RTK endocytosis regulators should be seen in the context of what is already
know about RTK signaling and regulation. Signaling from RTKs is initiated upon
transphosphorylation of activating tyrosines and docking tyrosines, the latter
generating binding sites for PTB and SH2 domain proteins. Receptor activation is
elicited by binding of activating ligand but can also occur if two receptor
molecules contact each other productively for other reasons. The likelihood of
ligand-independent activation depends on receptor density, and hence
overexpressed receptors may have ligand-independent activity in addition to
responding more strongly to ligands. Inactivation of receptors is therefore
critical for proper signaling in the cell. Phosphatases inactivate receptors by
catalyzing the reverse reaction of the activation. Phosphatases are very
abundant in cells and may be constitutively active. Local inactivation of
phosphatases is one mechanism that can lead to spreading of an initially
localized RTK signal. In
addition, signaling can be inactivated by endocytosis, which leads to
degradation of activated receptors, stimulated by molecules such as Cbl and
Sprint/RIN1 and at a later step by Hrs (Jekely, 2005).
Most studies of induced endocytosis, in order
to give maximal experimental resolution, have been done in tissue culture cells
with acute stimulation by high levels of ligand. In tissues, which have steady
and modest levels of ligand and a complex, multicellular environment, the role
of endocytosis in RTK regulation is less well understood. For example, Egfr
signaling is mildly increased in Cbl mutant follicle cell clones,
but so mild that even
in Cbl, sprint double mutant follicle cells, there are no detectable
changes in levels of Egfr, Pvr, or phospho-tyrosine. However, the effects on
border cell migration are striking. Receptor proteins do turn over in the tissue
and at least some of this turnover is blocked in Hrs mutant cells.
However, under physiological conditions in the ovary, the Hrs-dependent
degradation is not dependent on ligand and is not required for
guided migration. These results show that the physiological role of
Cbl and Sprint in border cell guidance is not to control receptor degradation
and/or to turn off signaling, but instead to keep the signal localized (Jekely, 2005).
It is
becoming appreciated that endocytosis of signaling receptors is not simply a
matter of signal attenuation and receptor removal. It was found that RTK
endocytosis differentially affected signaling through different pathways,
suggesting (1) that signaling can happen in different compartments and (2)
that the process of endocytosis could be used to differentially regulate
signaling outcomes. For TGF-β signaling, the process of endocytosis actively brings receptors to internal signaling. This study suggests a third
role for early aspects of receptor endocytosis in signaling: to keep
active signaling complexes localized in the plane of the membrane. This activity
prevents signaling from becoming uniform and therefore uninformative about the
spatial distribution of the ligand (Jekely, 2005).
How do Cbl and Sprint spatially restrict
signaling? They may prevent signaling from becoming delocalized by restricting
lateral movement of activated receptors or lateral spread of RTK activation.
Microdomains of
active RTKs on the plasma membrane or in endocytic pits could maintain activity,
whereas they would be inactivated at other
places by ubiquitous phosphatases. Alternatively, recycling of activated
receptors to new regions of the cell membrane could delocalize signaling.
Normally, this recycling might be prevented by Cbl
and Sprint activity by routing active RTKs to degradation via the proper
endosome compartment (without requiring Hrs). For these two scenarios, however,
it is not obvious why physically blocking endocytosis (Shibire
dominant-negative) should also delocalize signaling. Shibire/dynamin is a
general effector of endocytosis (required for cell viability), and interfering
with it therefore is a more blunt tool than manipulating Cbl and Sprint or
mutating Pvr. But the effects are unambiguous. This leads to a third
hypothesis, whereby endocytosis of active RTKs allows their redelivery or
recycling to regions of higher signaling.
Endocytosis and plasma membrane redelivery of active proteins contributes to
polarization in yeast, another case of controlling spatial information. Obviously,
further analysis will be needed to fully explore these cellular mechanisms in
vivo. In any case, at sufficiently high level of receptor expression and
activation, the regulatory mechanism may collapse. Indeed, when a C-terminally
tagged Egfr was expressed at extremely high levels in border cells, migration
and phospho-tyrosine staining were perturbed in a manner similar to what was
observed for Cbl mutant clones. Like many regulatory
mechanisms, the spatial restriction imposed by Cbl and Sprint works effectively
within a certain range of input, emphasizing the need for understanding the
mechanism of regulation at a physiological range in vivo (Jekely, 2005).
The role of early
endocytosis regulators in spatial signal regulation described in this study is clearly
physiologically relevant. But how general might it be? The regulation is likely
to be relevant when RTKs are used for spatially resolved signaling. RTKs can act
as canonical guidance receptors to detect specific ligands. In addition,
mammalian Cbl is required for integrin-dependent migration of macrophages and
osteoclasts in vitro and in vivo.
This requirement was suggested to reflect an active signaling role of
Cbl downstream of integrins, but it could also reflect a role for Cbl in
localizing signaling analogous to what has been seen in border cells. There is
ample evidence for cross-talk between integrins and RTKs, which in
turn are regulated by Cbl. That RTKs can be activated in the absence of cognate
ligand by high receptor density or by cross-talk from other pathways such as
integrins might seem at odds with their serving as guidance receptors. But with
effective regulatory mechanisms to maintain localized signaling, this excitable
signaling system may help migrating cells obtain sufficient sensitivity to read
guidance cues over a large dynamic range (Jekely, 2005).
A key issue in guidance signaling is
that migrating cells must achieve a polarized output despite having to respond,
over a large dynamic range, to subtle concentration differences of an attractant
or repellant from one end of the cell to the other. One way to achieve this is
to amplify the initial signal difference between stimulation of receptors at the
front and back of the cell. The use of PI3 kinase and PTEN phosphatase, two
antagonistic enzymes that are reciprocally regulated in Dictyostelium
chemotaxis, may be an example of this. Alternatively, guidance cues may
simply bias a separate, preexisting intrinsic polarity in the migrating cells. Finally,
guidance signaling and intrinsic polarity may interact dynamically to reinforce
one another during directed migration. The net outcome is a robust difference
between the front and the back of the cell, allowing migration. It is
interesting to consider that RTK endocytosis may also function to enhance the
difference between signaling in the front and back of migratory border cells.
Although the gradient of RTK ligands around border cells can not be measured, it
is very unlikely to be as steep as the observed difference in phospho-tyrosine
staining. Enhancement of a signaling differential can occur at different levels.
Binding of Cbl and Sprint to activated RTKs and recruitment to membrane
subdomains may effectively concentrate activated receptors. Due to the density
dependence and positive feedback in RTK activation, local activity will then be
increased, whereas global inactivation by phosphatases could ensure that
signaling is reduced elsewhere. It is suggested that spatial
organization of signaling may be controlled by endocytosis and redelivery of
active receptors. Such active turnover processes can be used as an effective
mechanism to increase signaling differentials within a cell, leading even to
spontaneous, or self-organized, polarity. A distinct cell front and cell
rear and hence short-term productive migration is often seen in migrating cells
even without perception of localized guidance cues, indicating intrinsic
polarity. Most migrating cells may need to integrate intrinsic polarity and
external guidance. Using a regulatory principle that can produce both intrinsic
polarity and local response to guidance cues would provide an elegant means to
achieve this (Jekely, 2005).
Fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor have been identified and characterized. The gene encoding the ligand PDGF/VEGF factor 1 (Pvf1) has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The gene encoding the receptor, PDGF- and VEGF-receptor related (Pvr), has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic hematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the Pvr gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signaling system may regulate the migration of the Drosophila embryonic hemocyte precursor cells (Heino, 2001).
Northern blot analyses of total RNA from various developmental stages using a probe recognizing the intracellular part of all splice variants of Pvr has revealed a single major mRNA of about 5.5 kb. The transcript occurs in all embryonic stages and is also maternally supplied to the earliest embryonic stage. Zygotic Pvr mRNA is most abundant during later embryonic stages and nearly absent during larval development in the first and second instars, appearing again during the third instar. This expression persists during the pupal as well as adult stages (Heino, 2001).
In order to discriminate the various splice variants from each other, it was necessary to use isoform-specific probes, since the resolution of the agarose gel was not sufficient to clearly separate them. The major part of the RNA hybridization signal corresponds to the splice variant #1 as shown by comparison of the hybridizations with exon 6- and 7-specific probes. Small amounts of the SPV-2 and SPV-4 could be detected with the exon 7-specific and exon 5-specific probes, respectively. Both variants occurred with the same dynamic pattern as the dominant SPV-1. SPV-3 could not be assessed directly, but comparison of the hybridization signals obtained with the various probes shows that this variant is not expressed to any significant degree (Heino, 2001).
Pvr shows a very restricted temporal and spatial expression pattern during embryogenesis. At early stages (prior to stage 10), no signal is detected. Thereafter the gene is strongly expressed anteriorly in the presumptive head region. Later the expression becomes restricted to cells displaying a scattered distribution throughout the embryo. During stage 16, a sub-population of the cells expressing Pvr is clearly concentrated along the ventral midline. This pattern corresponds to the origin, migration and final distribution of the embryonic hemocytes (Heino, 2001).
Pvr transcripts are detected in mRNA from ovaries and from embryos. Pvr mRNA is detected in embryonic hemocytes and in the related tissue culture cells, Schneider cells. Endogenous Pvr protein was detected in Schneider cell extracts as an approximately 180 kDa protein, corresponding well to the predicted molecular weight of 170 kDa (Duchek, 2001b).
The embryonic expression pattern of Vegfr was determined by RNA in situ hybridization. Transcript is first detected in two bilaterally symmetric clusters of mesodermal cells in the head region of early stage 8 embryos (~4 hr after egg lay [AEL]). During the next 11 hr of development, these cells undergo a stereotyped series of migrations that disperse them throughout the embryo. The cells migrate out from the clusters in three directions. Some cells migrate anteriorly into the clypeolabrum, while others migrate ventrally toward the gnathal buds. The majority migrate posteriorly toward the caudal end (tail) of the germband-extended embryo, coursing between the amnioserosa and the yolk sac to reach its posterior margin. Once cells move past the posterior margin into the tail, they surround the hindgut opening and migrate along the ventral midline. By the beginning of stage 12 (~9 hr AEL), these migrations produce two populations of cells, one in the anterior scattered around their site of origin, the other in the posterior clustered around the hindgut and ventral midline. During stages 12-14 (~10-12 hr AEL), both populations migrate toward the middle of the embryo along three major routes: (1) the ventral midline (dorsal and ventral to the developing nerve cord); (2) the gut, and (3) the dorsal epidermis. Later, cells leave these paths and become uniformly distributed (Cho, 2002).
The positions and movements of most Vegfr-expressing cells resemble those of developing blood cells. Indeed, the Vegfr expression pattern is difficult to distinguish from that of the blood cell (plasmatocyte) markers Peroxidasin and Croquemort except that Vegfr turns on ~2 hr earlier. (It is not known if Vegfr is expressed in crystal cells, a minor subpopulation of hemocytes.) To confirm that Vegfr-expressing cells are hemocytes, serpentAS mutants, which lack blood cells, were examined. With the exception or three groups of cells, Vegfr-expressing cells are absent in srpAS embryos.
The three exceptions are (1) tracheal cells from stages 11-13; (2) a small group of cells near each tracheal visceral branch; and (3) a small group of ventral midline cells in each segment, possibly midline glia. In situ hybridization of fixed larvae indicated that larval blood cells also express Vegfr. Thus, Vegfr is expressed in most or all embryonic and larval blood cells throughout their development (Cho, 2002).
Blood cells play a crucial role in both morphogenetic and immunological processes in Drosophila, yet the factors regulating their proliferation remain largely unknown. In order to address this question, antibodies were raised against a tumorous blood cell line and an antigenic determinant was identified that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as PDGF- and VEGF-receptor related, a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, a dramatic increase was induced in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis (Munier, 2002).
Since Drosophila blood cells are difficult to obtain in sufficient numbers for immunization of mice, antibodies were raised against the cells of the tumorous blood cell line, mbn-2, which are functionally close to plasmatocytes. These cells phagocytose microorganisms and also synthesize and secrete antimicrobial peptides when exposed to bacteria or bacterial cell components. Live mbn-2 cells were injected into BALB/c mice and hybridomas were selected for their capacity to produce antibodies that recognize cell surface antigens on live mbn-2 cells. This recognition was monitored by flow cytometric analysis, and selected antibodies were tested for their abilities to affect the mitotic rate of mbn-2 cells. In these series, one antibody, 18G, strongly inhibited the proliferation of mbn-2 cells when added to the culture medium. The antiproliferative capacity of 18G was further ascertained by measuring its dose-dependent effect. Western blots performed with the 18G antibody on extracts of mbn-2 cells (under non-reducing conditions) revealed several protein bands of high molecular weight (>75 kDa;). Stained high-molecular-weight proteins were also detected in extracts of larval blood cells, as well as in Drosophila Schneider 2 (S2) cells (Munier, 2002).
The distribution of the 18G determinant on the different Drosophila hemocyte types was examined by immunocytochemistry. Wild-type and hopTum-l mutants were used for this purpose. hopTum-l is a Janus kinase (JAK) gain-of-function mutation resulting in an overproliferation of circulating blood cells, of which a large number are lamellocytes. Plasmatocytes were stained and lamellocytes were not. In hopTum-l mutants, the most strongly reacting cells were small rounded cells that correspond to circulating progenitor blood cells (prohemocytes). In wild-type larvae, the presence of prohemocytes is mostly restricted to lymph glands. Strong staining was observed on the prohemocytes of wild-type lymph glands. In hop-Tum-l lymph glands, the small rounded prohemocytes were also strongly stained, but other cells that have been described as lamellocytes but did not react with the antibody. Stained circulating crystal cells could not be observed due to their fragility. No staining was ever observed on larval tissues other than hemocytes, namely on fat body, muscles, imaginal discs, epidermal cells, brain or trachea (Munier, 2002).
Three protein bands positive for 18G staining in mbn-2 cell extracts were purified by immunoaffinity chromatography and subjected to Edman degradation. The N-terminal sequences obtained with each of the three protein bands were identical: VPLQQFSPDP. The Drosophila genome contains a single match to this sequence, namely in a gene encoding a homolog of mammalian receptors for PDGF and VEGF. Independent studies have identified expression of this gene in ovarian border cells and embryonic hemocytes, and the receptor is now referred to as PVR (PDGF/VEGF receptor). Pvr RNA interference experiments in S2 cells confirmed that 18G recognizes this receptor (Munier, 2002).
Attempts were made to study the role of PVR in Drosophila blood cell proliferation in vivo, using mutants or transgenes of Pvr. A transposon insertion (line PBc2195) in the 11th intron of the Pvr gene generates a null mutant that leads to embryonic lethality. Larvae heterozygous for this mutation (Pvrc2195/CyO) did not show defects in hemocyte counts. Overexpression of the Pvr cDNA by using a UAS-Pvr transgenic line crossed with either a daughterless-GAL4 line (for ubiquitous expression) or e33c-GAL4 line (for preferential lymph gland expression) is lethal at the embryonic stage. As an alternative, to establish the role of this receptor in hemocyte proliferation in larvae, the effect of ectopic expression of two of its putative ligands: [Pvf1 (CG7103) and Pvf2 (CG13780)] was analyzed (Munier, 2002).
UAS-Pvf1 and UAS-Pvf2 transgenic fly lines were generated and PVF1 and PVF2 expression was directed using daughterless-GAL4 and e33C-GAL4 drivers, and larval hemocytes were examined. PVF2 expression in both cases results in a dramatic increase (up to 300-fold) in the number of blood cells in third instar larvae and leads to pupal lethality. Apart from hemocytes and lymph glands, no proliferation was observed in other tissues. In contrast, overexpression of PVF1, using the same GAL4 drivers, results in a mild and variable effect on blood cell counts that does not exceed a 2-fold increase compared with controls. Overexpression of PVF1 also results in pupal lethality (Munier, 2002).
On average, the hemocytes in PVF2-overexpressing flies were noticeably smaller than in the wild type. All cells were reactive to the 18G antibody, indicating that they are prohemocytes or plasmatocytes. However, only a small percentage was able to phagocytose injected India ink, thus qualifying as fully mature plasmatocytes. Staining with anti-phosphohistone H3 antibody shows that a large number of circulating hemocytes were in the process of division, indicating that PVF2 stimulates proliferation rather than promotes cell survival. The vast majority of the PVF2-induced hemocytes are therefore to be considered as prohemocytes. Crystal cells were noticeably absent in w; UAS-Pvf2/+; e33C-GAL4/+ larvae (Munier, 2002).
Blood cells were counted in two lines with a transposon inserted in the Pvf2 gene, XPd2444 and PBc6947. These lines are homozygous viable and showed no obvious defects in blood cell counts. A Pvf1 loss-of-function mutant, Pvf11624, similarly showed no defect in larval blood cell counts. However, in this line, pupal lethality was observed (Munier, 2002).
Therefore, by using an antibody screening approach against a Drosophila blood cell line, the receptor tyrosine kinase PVR was identified as a marker of larval Drosophila hemocytes. This receptor is found on lymph gland prohemocytes and on the surface of mature circulating plasmatocytes/macrophages. The anti-PVR antibody inhibits thymidine incorporation in blood cell lines, whereas overexpression of one of its ligands, PVF2, induces the proliferation of hemocytes in vivo. These results strongly suggest that the PVF2/PVR couple is involved in hemocyte proliferation. PVR belongs to the PDGF/VEGF subfamily of RTKs. These receptors are characterized by an extracellular sequence composed of either five Ig domains (in c-Kit, Flt-3, c-Fms, PDGFRalpha and ß) or seven Ig domains (in VEGFRS: Flt1, KDR, Flt4) and a cytoplasmic split tyrosine kinase domain. In vertebrates, receptors of this RTK family function in both cell proliferation and cell migration (Munier, 2002).
PVR has recently been identified as a chemotactic receptor guiding cells to a source of PVF ligand, both in the context of ovarian border cell migration and in embryonic macrophage migration. The three PVFs (PVF1, PVF2 and PVF3) encoded by the Drosophila genome are thought to function redundantly during migration. The misexpression of a PVR ligand can disrupt the normal migration of border cells (i.e., PVF1 misexpression) or embryonic hemocytes (i.e., PVF2), but the removal of a single ligand is insufficient to block the migration process. A similar misexpression strategy was used to study larval hemocyte proliferation, with markedly different outcomes depending on the misexpressed ligand. This study clearly establishes that PVF2, and not PVF1, promotes hemocyte proliferation. More complex to explain is the complete absence of crystal cells in larvae overexpressing PVF2. This phenotype could result from the persistence of PVR-positive prohemocytes in a constant mitotic state by PVF2 stimulation, preventing their differentiation into crystal cells. Finally, the absence of any abnormal blood cell phenotype in transposon insertion lines of the Pvf2 gene implies that PVF2 is sufficient, but not absolutely required, in hemocyte proliferation (Munier, 2002).
The overexpression of PVF1 or PVF2 results in lethality during pupal development. In the case of PVF2, this phenotype is attributed to the enormous amount of blood cells that could disturb overall physiology of the larvae instead of metamorphosis per se. However, PVF1 misexpression reveals a more complex role for the PVR pathway in metamorphosis, possibly by disrupting the homing of hemocytes to, and/or engulfment of, larval apoptotic tissue. PVF1 seems important in metamorphosis, because a loss-of-function allele in the gene (Pvf11624) results in 40%-60% pupal lethality but has wild-type hemocyte numbers (Munier, 2002).
In vertebrates, hematopoietic stem cells are defined by their ability to self-renew and contribute to all lineages of mature blood cells. This self-renewal and differentiation are driven by numerous receptors that co-exist on the surface membrane of hematopoietic cells, among which are receptor tyrosine kinases of the PDGFR family (c-Kit, Flt-3, c-Fms, PDGFR). These factors act in combination with intracellular signal transducers. For example, it has been shown that JAK2 activation triggers extensive self-renewal of stem cells only if it is complemented by a second signal from c-Kit or Flt-3. Each of these proteins, JAK2, Flt-3 or c-Kit, alone is unable to sustain this activity. (Munier, 2002).
In Drosophila gain-of-function alleles in the JAK Hopscotch (e.g. hopTum-l) cause overproliferation of hemocytes. This report raises the obvious question as to what kind of interconnection exists between pathway(s) activated by PVR and the JAK/STAT pathway itself. Some cross-talk could occur, such as phosphorylation of STAT by PVF2-induced PVR activation. In mammals, for instance, PDGFR can directly activate some STATs. Conversely, evidence exists that JAK can activate the D-raf/D-MEK/MAP kinase pathway, one that is frequently activated by receptor tyrosine kinases. As is the case for PVF2, however, neither JAK nor STAT seem absolutely required for blood cell proliferation. Indeed, in loss-of-function mutants of hop or stat that permit larval development, blood cell counts are normal. This leaves open the possibility that upstream components of the JAK/STAT pathway, e.g., the receptor Domeless (DOME) and its ligand Unpaired, could act in synergy with the PVF2/PVR pathway. Both DOME and Upd are implicated in embryonic pair-rule gene expression, but their role in hematopoeisis awaits investigation (Munier, 2002).
In summary, the data indicate that PVR integrates two functions shared by mammalian receptors of the same subfamily. Like its mammalian VEGFR homologs (Flt1, KDR and Flt4), it regulates cell migration; and like c-Kit, Flt-3, c-Fms and most PDGFRs, it is implicated in the control of blood cell proliferation. In the light of the importance of hemocytes in development and in the innate immune response, it would be highly relevant to investigate further the interaction between PVFs, PVR, the JAK/STAT pathway and the downstream mitogenic factors that they induce (Munier, 2002).
Epithelial tissue functions depend largely on a polarized organization of the individual cells. The roles of the Drosophila PDGF/VEGF receptor (PVR) in polarized epithelial cells were examined, with specific emphasis on the wing disc epithelium. Although the receptor is broadly distributed in this tissue, two of its ligands, PVF1 and PVF3 are specifically deposited within the apical extracellular space, implying that polarized apical activation of the receptor takes place. The apical localization of the ligands involves a specialized secretion pathway. Clones for null alleles of Pvr or expression of RNAi constructs showed no phenotypes in the wing disc or pupal wing, suggesting that Pvr plays a redundant role in this tissue. However, when uniform expression of a constitutively dimerizing receptor was induced, loss of epithelial polarity, formation of multiple adherens and septate junctions, and tumorous growth were observed in the wing disc. Elevation of the level of full-length PVR also gave rise to prominent phenotypes, characterized by higher levels of actin microfilaments at the basolateral areas of the cells and irregular folding of the tissue. Together, these results suggest that polarized PVR activation is necessary for the proper organization of the wing disc epithelium, by regulating the apical assembly of the actin cytoskeleton (Rosin, 2004).
Examination of PVR protein has revealed a broad expression in epithelial
tissues in the embryo from stage 14, and in the imaginal discs. PVR expression is not confined along the apicobasal axis of the cells. In contrast to the uniform distribution of the receptor, there is restricted apical localization of the ligands PVF1 and PVF3 within the wing disc epithelium (Rosin, 2004).
The mechanism responsible for the apical accumulation of PVF1 and PVF3 in the wing disc is intriguing. Since cell junctions are likely to form barriers that can not be bypassed by exogenous ligand, apical accumulation may imply preferential secretion of PVR ligands at the apical compartment. It is possible that PVF1 and PVF3 are targeted to vesicles that are specifically marked for secretion at the apical surface. The observation that PVF3 overexpression compromises the secretion of PVF1 but not that of sGFP, supports such a possibility. The presence of distinct secretory vesicles that are targeted to apical versus basolateral compartments has been previously established (Rosin, 2004).
What further interactions do PVF1 and PVF3 undergo, once secreted to the apical extracellular compartment? One possible interaction involves binding to heparan-sulfate proteoglycans on the cell surface. The vertebrate VEGF proteins have a defined heparin-binding domain at the C terminus that is distinct from the receptor-binding moiety. The
equivalent C-terminal domain of PVF1 does not show a distinct homology to the heparin-binding domain of VEGF. However this study shows that PVF1 secreted by S2 cells can bind heparin beads. To examine if PVF1 is trapped on the cell surface following secretion, marked clones of cells overexpressing PVF1 were created. The ligand is uniformly redistributed along the entire apical surface, including the surface of cells not secreting the ligand. Although the ligand is capable of spreading readily within the apical plane, it is incapable of crossing the cell junctions, and is thus excluded from the basolateral extracellular compartment (Rosin, 2004).
Accumulation of PVF1 and PVF3 at the extracellular apical compartment
implies that the PVR receptor is activated in a polarized fashion. Does such an apically polarized pattern of activation play a role in shaping the wing disc epithelium? The most direct way to examine PVR function in the wing disc is to generate clones for null Pvr alleles, and follow their phenotype. The Pvr-mutant clones are similar in size to their wild-type twins, and within the clones no aberrant morphology or
misorganization of actin was detected. It is thus concluded that PVR has a
redundant role in the wing. Nevertheless, a series of dramatic wing phenotypes is induced following expression of various PVR constructs. This analysis leads to a proposal that these phenotypes represent gain-of-function circumstances following inappropriate activation of PVR on the basolateral side of the wing disc epithelium (Rosin, 2004).
This interpretation was suggested by the dramatic effects of
non-restricted and constitutive receptor activation, achieved by expression of lambdaPVR in the wing disc epithelium. The epithelium lost its polarity, multiple cell layers were generated, and giant tumorous discs were formed. Ectopic accumulation of F-actin around the circumference of the cells was observed, and corroborated by the identification of multiple adherens junctions in EM images (Rosin, 2004).
The phenotype created by expressing lambdaPVR in the wing disc is
reminiscent of the phenotype described for loss of the septate junction
proteins DLG and Scribbled, as well the LGL protein. It is believed that the alterations in cell polarity following lambdaPVR expression are less severe than the dlg, scribble or lgl mutant phenotypes. Although excess adherens junctions are established and the septate junctions are mislocalized, the LGL protein, which requires intact septate junctions for its insertion into the membrane, is found associated with the membrane in wing discs expressing lambdaPVR. The tumorous growth of the cells is believed to be a secondary consequence of the loss of polarity, which may lead to impairment of cell-cell communication (Rosin, 2004).
The consequences of misexpressing full-length PVR were examined. Significantly higher levels of F-actin were noticed in the basolateral area of the cells expressing PVR, while the level of actin monomers was lower. This indicates that PVR has a localized effect on actin polymerization, rather than a general role in actin monomer synthesis. Elevation in Profilin (Chickadee) protein levels was also noticed. Profilin binds actin monomers in a way
that inhibits nucleation and elongation of pointed ends but promotes rapid
elongation of uncapped barbed ends, leading to depletion of the actin monomer pool (Rosin, 2004).
Overexpression of the ligands alone did not lead to any phenotype, while even mild overexpression of the receptor resulted in pronounced phenotypes. Moreover, these phenotypes were strongly enhanced by elevating the levels of PVF1 in the apical domain. There are two possible explanations for the overexpression phenotype. The overall levels of receptor activation may be important. The receptor could be present in limited amounts, so that increasing its levels allows more ligand at the apical side to bind and activate receptors. Alternatively, polarized activation of the receptor that normally takes place is disturbed, because of redistribution of the ligand. This may happen by recycling of the ligand-bound receptor inside the cells. The fact that elevation in PVR levels resulted in basolateral polymerization of actin, while the ligand is normally found on the apical side supports this possibility. Mislocalized activation of the receptor may also take place because of spontaneous dimerization caused by the higher levels of the receptor (Rosin, 2004).
It is interesting to note that while lambdaPVR, a constitutively dimerized form of PVR, gave rise to a dramatic phenotype when expressed in the wing disc or the follicular epithelium, no apparent phenotypes were observed following expression in the embryonic ectoderm or the eye disc. Some of the intracellular elements that may be essential for relaying the signals resulting from PVR activation could thus be expressed or active only in a restricted set of tissues (Rosin, 2004).
In the embryonic ectoderm and eye disc where lambdaPVR was inactive, apical accumulation of PVF1 was not seen. The correlation between the
capacity of the wing epithelium to localize the ligands apically, on the one hand, and to respond to uniform PVR activation, on the other, strengthens the notion that apical activation of PVR is instructive in this tissue (Rosin, 2004).
What can the ectopic phenotypes teach us with regard to the normal
downstream responses to PVR activation in the wing epithelium? The primary
defect upon overexpression of PVR is misorganization of the actin cytoskeleton at the basolateral side. In addition, expression of the constitutively active receptor results in multiple adherens junctions. It is thus suggested that apically restricted PVR activation provides signals that facilitate the formation of F-actin at the adherens junctions. This role is reminiscent of the activity of PVR in the border cells of the ovary, where polarized activation by PVF1, expressed in the oocyte, participates in guiding the migration of the border cells. It is tempting to suggest that polarized PVR activation regulates migration or cell polarity, using a common set of intracellular responses leading to localized actin polymerization (Rosin, 2004).
The Drosophila adult male terminalia originate from the genital disc. During the pupal stages, the external parts of terminalia evert from two ventral stalks; the everted left and right dorsal halves fuse at the dorsal midline. At the same time the male terminalia perform a 360o clockwise rotation. Several mutations are known to affect the rotation of the male terminalia, while none is known to affect dorsal closure. This study shows that the Pvf1 gene, encoding one of the three Drosophila homologues of the mammalian VEGF/PDGF growth factors, is required for both processes. Males either mutant for Pvf1 or bearing a dominant negative form of Pvr, the PVF receptor, do not complete either rotation or dorsal closure. Pvf1 expression in the genital disc is restricted to the A8 cells. However, PVF1/PVR signaling influences A8, A9 and A10 cells, suggesting that the PVF1 protein diffuses from its source. Flies hemizygous for the apoptotic genes hid, reaper and grim, or mutant for puckered which encodes a phosphatase that down-regulates the n-Jun-N terminal kinase pathway, lead to the same phenotypes as mutations in PVF1/PVR. These results indicate that PVF1/PVR signaling functions not only in apoptotic phenomena but are also required during rotation and dorsal closure of the Drosophila male genital disc (Mac̀as, 2004).
This demonstrates that either mutations at Pvf1 or the
expression of a dominant negative form of its receptor,
PvrDN, result in various degrees of male rotated terminalia and
failure of dorsal closure. These observations indicate that the
PVF1/PVR pathway is relevant in these morphogenetic processes.
Although the Pvf1 gene is only expressed in a subset of cells from
the segment A8, reduction or abolition of PVF1/PVR signaling
affects the normal development of all terminalia precursors (A8, A9
and A10). Interestingly, mutations in the Abd-B m function, which
affect only the A8 segment have a phenotype of rotated terminalia. Thus, these results highlight the importance of the A8 segment in this process. It is proposed that A8 cells affect the development of structures originated from A9 and
A10 through the activity of the PVF1 protein diffusing from A8.
Although the data concern transcript expression, Rosin
(2004), demonstrated that PVF1 is capable of extensive lateral
diffusion, so it has the properties of a long range signaling molecule.
PVFs could form homo and heterodimers, what opens the
possibility of different effects in the binding responses of the
receptor. McDonald (2003), observed that homodimers are
not equivalent, because PVF1 seems to be the relevant signal for
the migration of border cells and Bruncker (2004), describe
two function for PVR in the embryonic hemocytes, suggesting a
diversity of functions. Other PVFs have not been examined, and
although some partial redundancy was observed, it will be necessary
to separate PVF individual or associated functions (Mac̀as, 2004).
Indirect evidence was obtained about where the PVR receptor
is activated or expressed. First, evidence was obtained by recognition of factors that mediate the activity of the PVF1/PVR signaling mechanism (i.e.
dpERK), whose expression is located at the periphery of the
group of cells expressing Pvf1. Second, it was shown that blocking PVF1 activity using PvrDN and overexpressing Pvf1 results in stronger effects in the engrailed domain where Pvf1 is not expressed. These findings provide additional evidence that there are specific domains for
ligand expression and for responsive cells. In the ovary Pvf1 is
expressed in the ovule while Pvr is expressed in the follicle cells,
the importance of this non-overlapping domains is reflected by the
fact that overexpression of a constitutive active form of the
receptor (lambda Pvr) produces the same phenotype of its lack of
function. In the wing disc Rosin (2004)
observed that the restrictions in the activity are regulated by a
polarized secretion of the ligand in the apical membrane (Mac̀as, 2004).
Mutations in the pro-apoptotic gene hid have been shown to
affect male terminalia rotation, although
this phenotype was observed in trans heterozygotes for Df(3L)H99,
which includes the three pro-apoptotic genes hid, rpr and grim. Trans heterozygotes for hid mutations are of
wildtype phenotype, indicating that the rotated phenotype over
deficiency is not only due to hid but to the haploinsufficiency of one
or the two other genes. The result that preventing cell death with
p35 leads to miss rotation and split dorsal is also consistent with an
involvement of apoptosis in these processes (Mac̀as, 2004).
Additionally, it was shown that overexpressing puc results in the
same phenotypes as PVF1/PVR and reduction of apoptosis;
lowering puc rescues the rotated terminalia defects observed in
DfH99/+ males. The level of puc is considered as indicative of the
JNK pathway activity, so these experiments suggest that JNK promotes
apoptosis, probably by upregulating hid (Mac̀as, 2004).
The fact that alterations in the PVF1/PVR pathway and in JNK/
apoptosis give rise to similar phenotypes suggests a functional
link between these two pathways. The penetrance of the phenotypes of Pvf1 mutations in the terminalia increases when they are additionally heterozygous for
Df(3L)H99. This increase is nonadditive, suggesting PVF1/PVR
and the apoptotic machinery affect the same aspect of the
process. The overexpression of Pvf1 ectopically activates puc
and impedes the normal rotation and closure. This activation
would down-regulate the JNK apoptotic pathway, thus reducing apoptosis and giving rise to the terminalia phenotype, but since the JNK pathway is a transcriptional
activator of puc, this result
opens up the possibility that Pvf1 ectopically activates JNK rather
than puc (Mac̀as, 2004).
Apoptosis is necessary for terminalia rotation and dorsal closure
and these results indicate that it is mediated
by JNK activity. PVF1/PVR is also affecting these processes and
the data suggest that PVF1/PVR may also affect JNK-mediated
apoptosis. It is not clear however, whether all these elements act
on the same developmental cascade (Mac̀as, 2004).
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).
The Drosophila embryonic salivary gland is a migrating tissue that undergoes a stereotypic pattern of migration into the embryo. This study demonstrates that the migratory path of the salivary gland requires the PDGF/VEGF pathway. The PDGF/VEGF receptor, Pvr, is strongly expressed in the salivary glands, and Pvr mutations cause abnormal ventral curving of the glands, suggesting that Pvr is involved in gland migration. Although the Pvr ligands, Pvf1 and Pvf2, have distinct expression patterns in the Drosophila embryo, mutations for either one of the ligands result in salivary gland migration defects similar to those seen in embryos that lack Pvr. Rescue experiments indicate that the PDGF/VEGF pathway functions autonomously in the salivary gland. The results of this study demonstrate that the Drosophila PDGF/VEGF pathway is essential for proper positioning of the salivary glands (Harris, 2007).
Cell migration is an essential part of the development and function of many cell types in all multicellular organisms. Guidance by external spatial cues directs a migrating cell or tissue to maintain an appropriate migratory path within an organism and ultimately reach the correct target. There are many examples of this, including immune cells that receive chemical gradient cues throughout development, as well as during their lifetime as pathogen fighting cells, neurons that receive cues promoting axon guidance, the multistep migration of the primordial germ cells and migration of the border cells in the Drosophila ovary (Harris, 2007).
The embryonic development of the Drosophila salivary glands provides a good system to study guided cell migration. The salivary glands consist of two cell types: gland cells and duct cells, which are specified on the ventral surface of parasegment 2. During stage 11, the circular salivary placodes form and are visible as two groups of cells on either side of the ventral midline. The placodes are separated ventrally by cells that will give rise to the salivary ducts. After specification, the salivary placodes begin to invaginate into the embryo. When the salivary glands reach the visceral mesoderm, the glands turn and begin posterior migration. The glands are completely internalized by stage 13 and lie parallel to the anteroposterior axis of the embryo. This posterior migration is a heavily regulated process involving attractive and repulsive cues and complex tissue-tissue interactions that are just beginning to be understood. Recent work on these cues has revealed startling similarities between salivary gland migration and axonal development (Harris, 2007).
This study characterized the role in salivary gland development of Pvr, the gene coding for the single Drosophila homolog of the mammalian PDGF/VEGF receptors. Previous studies have shown that Pvr is needed for border cell migration, hemocyte migration and survival, thorax closure during metamorphosis, and the rotation and dorsal closure of the male terminalia. These processes involve concerted morphogenetic cell movements which are disrupted in Pvr mutants. This study reports that Pvr is expressed in Drosophila embryonic salivary glands and that mutations in Pvr disrupt the concerted migration of the salivary glands. Furthermore, at least two of the Pvf ligands, Pvf1 and Pvf2 are required for this migration (Harris, 2007).
In Drosophila embryos the Pvr receptor is expressed in the hemocytes where it is necessary for cell survival and for migration of the hemocytes throughout the embryo. Another site of embryonic Pvr expression is the developing salivary gland. Salivary expression of Pvr mRNA is strongest at stage 11 of embryonic development, when salivary gland cells are still situated on the surface of the embryo as circular placodes. Transcript levels steadily decrease through stage 12, during which time the placode cells invaginate. At stage 13 Pvr transcripts are practically undetectable. PVR protein is detected in the gland beginning at stage 12 and is localized to the cell membrane (Harris, 2007).
Three genes in the Drosophila genome code for Pvr ligands: Pvf1, Pvf2, and Pvf3. Both Pvf2 and Pvf3 are expressed in the ventral midline, where they are thought to act in a partially redundant manner as attractive cues for hemocytes migrating out of the head. Previous studies have shown that Pvf2 and Pvf3 share more than just a similar expression pattern. These genes are located only 16 kb apart and may have been generated by recent gene duplication. Sequence similarities indicate that they are likely to be functionally similar to each other as well. In contrast, Pvf1 contains unique, cysteine-rich CXCXC motifs not found in the other two ligands, and it has a distinct expression pattern. The developing salivary gland is the strongest site of Pvf1 expression, beginning at stage 12 and persisting through stage 17. Interestingly, Pvf1 protein is expressed at highest levels in the cells near the tip of the gland that are the most actively involved in migration. Pvf1 is not expressed in