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).
Border cell migration during Drosophila oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. This study shows that the metastasis suppressor gene homolog Nm23/awd (abnormal wing discs) is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin (Shibire or Shi). The functional relationship between awd and shi is highly specific and almost exclusive in the endocytic pathway. The functional relationship between Nm23/Awd and dynamin has prompted the suggestion that Nm23/Awd is a GTP supplier for dynamin, a GTPase. Nonetheless, the putative antimetastasis activity of Nm23/Awd has never been demonstrated in a physiologically relevant metastasis or epithelial to mesenchymal transition model. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion (Nallamothu, 2008).
This report describes a novel role of a negative regulator of directional migration in border cells. Specifically, the significance of developmentally regulated loss of Awd expression in border cells during their active migratory phase was studied. Ectopic expression of Awd effectively blocks border cell movement, suggesting that Awd is involved in modulating the directional movement of the border cell complex. Conversely, a high level of Awd expression was observed in the nonmigrating follicle cells, possibly promoting rapid turnover of surface receptors to prevent ectopic cell migration. Indeed, loss of awd in these cells and in premigratory border cells resulted in up-regulation of Pvr and stalling of border cells, consistent with the phenotype of overactive Pvr signaling reported previously observation of overexpression of wild-type Pvr. The results show that the function of Awd is to promote down-regulation of the surface receptors such as Pvr and Dome, in collaboration with Shi/dynamin, thereby regulating the chemotactic signal strength. Although the function of Awd has been linked to dynamin (Dammai, 2003; Krishnan, 2001), this report is the first on the relevance of the Nm23/Awd antimetastasis function in an analogous developmental model. This study has demonstrated that border cell migration is stalled by both ectopic expression of Awd in the migrating cells and knockdown of Awd in premigrating cells, although through opposite consequences of reducing and increasing Pvr expression, respectively. This is consistent with the published finding that both loss of function and gain of function in Pvr signaling can disrupt border cell migration, due to loss of chemotactic signal or overwhelming signal, respectively. It was proposed and subsequently demonstrated by time-lapse microscopy that border cells that are oversaturated with Pvf signaling spin around without moving forward, consistent with the overborne, nondirectional chemotactic signaling response; while pvr loss-of-function border cells do not move. That is, although the phenotypic outcomes are the same, the cellular behaviors of the two genetic conditions are just the opposite (Nallamothu, 2008).
At this time, the cellular events that precisely down-regulate Awd expression in migrating border cells remain unknown. However, the observations suggest that the regulatory mechanism, besides potential transcriptional regulation, could at least in part be posttranscriptional. For example, the slbo-GAL4 driver can usually induce very high levels of ectopic expression, as evidenced by the expression of UAS-{lambda}pvr in this study. However, with UAS-awd (without the endogenous 3' untranslated region), it was possible to achieve at best a level equal to the endogenous one in nearby follicle cells and very often much lower (Nallamothu, 2008).
The histidine-dependent phosphotransferase activity of Nm23/Awd has functional correlation with the production and usage of GTP and the Awd-GTP link is worth noting since dynamin is a GTPase. In a classic study to identify components of eye color pathway, one peculiar, otherwise healthy mutant caused dominant lethality in the viable eye color prune null mutant background (Sturtevant, 1956). This dominant conditional lethal allele was named Killer-of-prune (K-pn) and turned out to be a missense mutant allele of awd. This is highly interesting because the Drosophila eye pigmentation is determined by pteridines that is also a precursor of essential enzyme cofactors. The rate-limiting enzyme in pteridine biosynthesis is GTP cyclohydrolase, which uses substrate GTP to generate dihydroneopterin triphosphate. It was suggested that the Prune protein, which contains pyrophosphatase activity, stabilizes or promotes Nm23/Awd multimeric protein activity by channeling the phosphate. It is possible that Awd and Prune proteins together form a relay system for generating GTP. Therefore, the K-pn mutation of awd in the prune mutant background renders the phosphate transfer function of the Prune-Awd protein complex even less stable. Indeed, among the myriad of interacting proteins of Nm23 in mammalian cells, many are related directly or indirectly to the GTPases, such as Arf6, TIAM1 (a guanine exchange factor for Rac), Lbc (a guanine exchange factor for Rho), and Rad. Whether or not these GTPase-related functions hold true requires further in vivo investigation. Recently, the lysophosphatidic acid receptor EDG2 was found to be overexpressed in Nm23-H1 mutant metastatic breast cancer cells, which can account for the metastatic activity of this cell line. However, whether the up-regulation is a direct or downstream effect of Nm23 loss of function is not clear. It therefore remained to be determined whether the similar receptor down-regulation mechanism by Awd observed in this report is applicable to EDG2 regulation (Nallamothu, 2008).
It should be noted, however, that although the observed genetic interaction between awd and shi suggests that Awd may promote the endocytic activity of Shi/dynamin, it is formally possible that Awd may promote protein turnover that is downstream of the initial endocytic event. On this note, it is also worth considering other activities of Nm23/Awd. The results showed that substitution of the active-site histidine residue that is critical for the nucleoside diphosphate kinase activity could not stall border cell migration. This is consistent with previous finding that this residue is required for rescuing the enhancer of shi phenotype (Krishnan, 2001). Curiously, this residue is not required for suppressing the in vitro motility (assayed by Boyden chamber) of the metastatic breast cancer cells. However, the histidine substitutions employed in the two systems are different (phenylalanine in human versus alanine in fly). It is therefore difficult at this time to draw a direct comparison. In contrast, human mutants that affect the histidine-dependent protein kinase activity failed to suppress the in vitro motility of the cancer cells. So far, very few Nm23 protein kinase targets have been identified and none verified in physiological settings. Nonetheless, the protein kinase activity may be of specific functional significance since the range of targets is likely limited, so that specific pathways that contribute to metastasis may be identified more readily. The border cell migration model describe here should be used in future studies to test the functions of Nm23/Awd based on the above-mentioned human mutations (Nallamothu, 2008).
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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 2004).
In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. This study shows the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerates as development proceeded, and completes a full 360° rotation. This acceleration is impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that form a ring shape and surround the male genitalia: the inner ring rotates with the genitalia and the outer ring rotates later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlates with the movement of the outer ring, and upregulation of the apoptotic signal increases the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame (Kuranaga, 2011).
To visualize the genitalia rotation in living animals, the His2Av-mRFP Drosophila line was used whose nuclei are ubiquitously marked by a fluorescent protein. The genital disc is a compound disc comprised of cells from three different embryonic segments: A8 (male eighth tergite), A9 (male primordium) and A10 (anal). During metamorphosis, the genital disc is partially everted, exposing its apical surface, and adopts a circular shape. The results captured the male genitalia undergoing a 360° clockwise rotation. Inhibiting apoptosis by expressing the baculovirus pan-caspase inhibitor p35 driven by engrailed-GAL4 (en-GAL4), which is expressed in the posterior compartment of each segment, results in genital mis-orientation at the adult stage (Kuranaga, 2011).
In flies expressing nuclear fluorescent protein driven by en-GAL4, it was observed that the posterior part of the A8 segment (A8p) formed a ring of cells surrounding the A9-A10 part of the disc. First, the images were recorded at a low resolution (10× objective lens) to measure the rotation speed accurately in control and p35-expressing flies, because long-term time-lapse imaging at a high resolution can cause photodamage, and thus alter pupal development. Most of the cells in the A8p that seem to disappear actually moved out of the plane of focus. The imaging results, the rotation started around 24 hours APF (after puparium formation) and stopped about 12 hours later. To confirm whether the mis-oriented genital phenotype in the caspase-inhibited flies was caused by incomplete rotation, the rotation was observed in flies expressing p35 under the en-GAL4 driver. In the p35-expressing flies, the rotation began, but it stopped before it was complete, after about 12 hours, i.e. with the same timing as in control flies. This suggested that the reduced caspase activation in A8p prevented the genitalia from completing the rotation, resulting in mis-oriented adult genitalia (Kuranaga, 2011).
To compare complete rotation with incomplete rotation, the rotation speed was calculated by measuring the angle (thetacontrol and thetap35) of the A9 genitalia every 30 minutes on time-lapse images. The normal rotation was composed of at least four steps: initiation, acceleration, deceleration and stopping. The velocity of rotation V=dtheta/dt was calculated by measuring theta as a function of time t. At first, the genitalia rotated at an average velocity (Vcontrol) of 7.67±3.72°/hour by 1 hour after initiation, then the rotation accelerated, with Vcontrol gradually increasing to 53.83±7.11°/hour by 7 hours after initiation. Interestingly, in the p35-expressing flies, the rotation normally started at 24 hours APF, and the average velocity (Vp35) from the initial rotation to 1 hour later was 7.45± 2.98°/hour, which was not significantly different from the normal rotation. However, the acceleration of the rotation in the p35-expressing flies was lower than normal, with Vp35 gradually increasing to 21.35±7.45°/hour at 5.5 hours after initiation. The first peak of the acceleration rate, which was defined as the initiation of rotation, was observed in the p35-expressing flies (ap35) and was the same as in the control flies (acontrol). However, the duration of the acceleration period was shorter in the p35-expressing flies. These data suggest a relationship between apoptosis and the acceleration of genitalia rotation (Kuranaga, 2011).
Next, the signaling mechanism(s) involved in the acceleration of genitalia rotation wee examined. The inhibition of JNK (c-Jun N-terminal kinase) and PVF (platelet vascular factor) signaling in male flies has been shown to result in mis-oriented adult male terminalia, and it has been hypothesized that the PVF/PVR (PVF receptor) may affect the genitalia rotation via JNK-mediated apoptosis (see Benitez, 2010). Consistent with previous reports, the acceleration of genitalia rotation was significantly impaired in flies expressing dominant-negative forms of JNK (JNK-DN) and PVR (PVR-DN). These data implied that caspase activation and JNK signaling contribute to driving the acceleration of genitalia rotation (Kuranaga, 2011).
To analyze how the genitalia accelerate their rotation, the movement of A8p was traced at the single-cell level. For this experiment, live imaging was performed at a high resolution (20× objective lens), which enabled the cells in A8p to be tracked at single-cell resolution. Cells that were neighbors of A9 rotated with A9, whereas cells located in the anterior half of A8p rotated later than A9. Based on this imaging, A8p was divided into two sheets, named A8pa (anterior of A8p) and A8pp (posterior of A8p). It was found that a part of the cells in A8p underwent apoptosis (Kuranaga, 2011).
To observe caspase activation in living animals, a FRET (fluorescence resonance energy transfer)-based indicator, SCAT3 (sensor for activated caspases based on FRET) was generated. To precisely evaluate apoptosis, a nuclear localization signal-tagged SCAT3 (nls-SCAT3; UAS-nls-ECFP-venus) was used. The nls-SCAT3 signal was clearly observed in A8p. Cells exhibiting high caspase activity were extruded into the body cavity and disappeared, consistent with their apoptotic death and engulfment by circulating hemocytes. Each cell was tracked in the A8p region during the first half of the rotation, and it was found that at least three types of cellular behavior were observed: (1) cells located in A8pp moved with A9, (2) cells underwent apoptosis and (3) cells located in A8pa rotated later (Kuranaga, 2011).
Thus, to observe the behavior of the cells in A8pa, Abdominal B (AbdB) was used as an A8 marker. AbdB is a homeotic gene that is required for the correct development of the genital disc, and AbdB-GAL4LDN is expressed in the segment A8 (in A8a and A8p) of the genital disc during the 3rd instar larval stage. At 24 hours APF, AbdB was expressed in A8 and formed a ring. Time-lapse images were taken, and unexpectedly it was found that most of the cells in the AbdB-expressing region underwent a 180° clockwise movement, suggesting that AbdB was not expressed in the A8pp region that moved 360° with A9. To determine the speed of the AbdB-expressing cells, three individual cells were traced in each fly, and the value of the turning angle of the cells (thetaAbdB) was calculated. The findings confirmed that the AbdB-expressing region moved halfway around. Although cells in the AbdB-expressing region moved only 180°, the A8pp (inner ring), which was encircled by the AbdB-expressing region (outer ring), still moved 360°. Furthermore, the imaging data indicated that the movement of the outer ring started 1-2 hours later than that of the A9 region, when the acceleration of the genitalia rotation occurred. These observations raise the possibility that the outer ring movement is related to the acceleration of the genitalia rotation (Kuranaga, 2011).
It was therefore considered that the outer ring movement was restricted in the p35-expressing flies, resulting in an incomplete genitalia rotation of about 180°, which mimics the movement of only the inner ring. To verify this possibility, the movement of the outer ring was examined in the p35-expressing flies (en-GAL4+UAS-p35). Although the inner ring rotated normally, the rotation of the outer ring was impaired in the p35-expressing flies. The turning angles were determined by tracing cells in the p35-expressing flies and it was found that thetap35 _inner increased, while the increase of thetap35 _outer was impaired. These data suggest that the A8 segment is composed of two independently regulated rings, and when apoptosis is inhibited, the inner ring can move only 180° with no outer ring movement, resulting in incomplete genitalia rotation (Kuranaga, 2011).
Thus, to determine whether apoptosis correlates with the outer ring movement, the apoptosis was quantified in A8pa every 10 minutes from 0-8 hours after the start of genitalia rotation. The frequency of apoptosis (Rapoptosis) was normalized to the total number of apoptotic cells in each individual. Pulsatile increases in Rapoptosis were observed, with peaks at 1, 2.5 and 4 hours after the start of genitalia rotation. To verify the participation of Rapoptosis in the initiation of outer ring movement, the acceleration rate of thetaAbdB (aAbdB) was calculated by measuring VAbdB as a function of time t, and Rapoptosis was compared with aAbdB. The starting time of outer ring movement was characterized by the early peaks of aAbdB. The analysis suggested that the aAbdB was related to the Rapoptosis, because aAbdB showed its first two peaks at about 1 and 2.5 hours after genitalia rotation started. To quantify these observations, the correlation was calculated between Rapoptosis and aAbdB. This analysis confirmed that there was a strong correlation between these parameters, because the correlation between aAbdB and Rapoptosis is approximately linear during this time. Therefore, these data implied a possible mechanism of apoptosis that facilitates the outer ring movement (Kuranaga, 2011).
To verify this possibility, whether the upregulation of apoptotic signals induces an increase in genitalia rotation speed was meastured. Because the expression of apoptotic genes using an en-GAL4 driver, which is expressed at the embryonic stage, is lethal, the TARGET system was used to control gene expression temporally. Flies were allowed to develop at 18°C until the head of the pupae had just everted, to inhibit gene expression. The pupae were then heat-shocked at 29°C for 12 hours to induce gene expression. Live imaging was performed at 22°C, after the heat shock. At this temperature, the genitalia rotation in the control flies was slower than in control flies bred at 25°C, because a low breeding temperature affects the rate of fly development, including genitalia rotation. Therefore, it was necessary in this experiment to compare the rotation speeds at the same temperature. The expression of reaper (rpr), a pro-apoptotic gene, using the TARGET system, showed that the upregulation of apoptotic signaling significantly increased the timing of acceleration and speed of genitalia rotation. These observations led to the proposal that the outer ring functions like a 'moving walkway' to accelerate the speed of the inner part of the structure, including the A9 genitalia, enabling genitalia to complete rotation within the appropriate developmental time window (Kuranaga, 2011).
According to these observations, it was found that apoptosis drives the movement of cell sheets during the morphogenesis of male terminalia. Further questions remain with regard to how apoptosis contributes to the cell sheet movement. A recent study indicated the possibility that local apoptosis acts as a brake release to regulate genitalia rotation, coupled with left-right determination (Suzanne, 2010). However, it has been reported that the cell shape change by apoptosis enables not only the extrusion of dying cells, but also the reorganization of the actin cytoskeleton in neighboring cells. Therefore, apoptosis could affect the behavior of neighboring cells, to act as a main driving force of the cell-sheet movement. Taken together, apoptosis may generally participate in the morphogenetic process of cell-sheet movement during morphogenesis (Kuranaga, 2011).
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 the ventral midline and has does not have a significant effect on embryonic hemocyte migration (Harris, 2007 and references therein).
The importance of Pvr and the Pvf ligands for salivary gland development was confirmed when Pvr mutant embryos were found to have salivary gland migration defects. Pvr null mutant embryos have salivary glands that curve abnormally toward the ventral surface of the embryo, instead of lying parallel to the A-P axis of the embryo as in wild-type embryos. In contrast to hemocytes, salivary gland survival, as tested by TUNEL staining, was not affected by Pvr mutations (Harris, 2007).
Mutations in the ligand genes Pvf1 or Pvf2 caused ventral curving similar to that in Pvr mutant embryos. In contrast, Pvf3EY09531, a P-element insertion located in the first intron of the Pvf3 gene, very infrequently affects embryonic gland positioning. This impenetrant phenotype may be due to the timing of Pvf3 expression, which occurs prior to salivary gland invagination and decreases during the time that the salivary gland migrates posteriorly. Alternatively, the EY09531 P-element insertion may be a weak, hypomorphic mutation that does not eliminate Pvf3 function (Harris, 2007).
Several factors required for salivary gland guidance and migration have already been identified. After the salivary gland contacts the VM and begins to move posteriorly within the embryo, the attractant Netrin and two repellents, Slit and Wnt4, guide the salivary glands. Netrin, which is expressed in the CNS and the visceral mesoderm, works as an attractant to maintain salivary gland positioning on the visceral mesoderm. At the same time, CNS expression of Slit and Wnt4 keeps the salivary glands away from the CNS and parallel to it. The early timing of the Pvr phenotype and its similarity to the slit and Wnt4 phenotypes suggest that PDGF/VEGF signaling is required at the same time as Netrin, Slit and Wnt4 signaling and might be required for the same process, salivary gland guidance. Near the end of salivary migration, a different signal-receptor pair, Wnt5 and Derailed, is required at the distal tip of the glands to mediate attachment to the longitudinal visceral mesoderm (Harris, 2007).
In addition to its role in the salivary gland, Pvr is essential for hemocyte migration throughout the embryo. During their migration hemocytes lay down components of the extracellular matrix needed by other cells for movement. For example, the extracellular matrix is indispensable for the process of ventral nerve cord condensation. In Pvr mutants, this condensation fails due to defects in hemocyte migration and extracellular matrix deposition. This extracellular matrix might also be important for salivary gland migration. Therefore, whether the salivary gland defects caused by mutations in Pvr and its ligands were autonomous to the salivary gland was investigated. When expression of a dominant negative allele of Pvr is driven in the salivary glands by fkh-GAL4, the glands curve ventrally as they do in Pvr mutants. In contrast, expression with hemocyte specific driver pxn-GAL4 results in near background levels of ventral curving, despite the inhibition of hemocyte migration. Thus, Pvr activity is autonomously required in the salivary glands. Neither proper hemocyte migration nor the extracellular matrix that hemocytes deposit is required for salivary gland migration. Furthermore, the gland migration defects in Pvr mutants are rescued in embryos carrying fkh-GAL4 and either UAS-Pvr or the constitutively active UAS-λPvr construct (Harris, 2007).
Previous studies in Drosophila have shown that the PVF ligands act as attractants. In the border cells, ectopic expression of PVF1 is sufficient to redirect border cells towards the site of expression. Similarly, PVF2 and PVF3 in the ventral midline act as attractants for migrating hemocytes. Ectopic expression of PVF2 using breathless-GAL4 has been shown to induce a chemotactic response to the new source of PVF expression from the embryonic hemocytes. The ventral midline expression of PVF2, along with its ventral curving, mutant phenotype, suggests that PVF2 might be acting as a repellent for the salivary gland, despite the fact that there is no previous indication of PVF ligands acting as repellents. However, ectopic expression of PVF2 in the visceral mesoderm was not sufficient to redirect the salivary glands away from the visceral mesoderm (Harris, 2007).
Overexpression of Pvf1 in the salivary glands results in ventrally curved glands similar to those seen in Pvr mutants. Similarly, misrouted glands result from salivary gland expression of Pvf2 and Pvf3. These results suggest that all three ligands may have the same or similar effects on the PVR receptor, despite their differing expression patterns and slightly different sequences. Interestingly, expression of the constitutively active receptor, λPVR in otherwise wild-type embryos results in salivary glands that are ventrally curved, similar to overexpression of the individual ligands in the gland. One possible explanation for these results is that the PVF ligands might be required for proper salivary gland positioning by regulating adhesion or another migratory event autonomous to the salivary gland (Harris, 2007).
Alternatively, both the salivary gland expressed PVF1 and ventral midline expressed PVF2 may be required for a directional response to be received, perhaps forming a heterodimer to activate the PVR receptor. This possibility is attractive since vertebrate studies have shown that heterodimers are formed between PDGF-A and PDGF-B that have a unique binding affinity for PDGF receptor subtypes that differs from the affinity of either homodimer. Furthermore, the PDGF-AB heterodimer is capable of activating different signal transduction pathways thus eliciting a different response on proliferation as well as gene expression compared to the PDGF-AA or PDGF-BB monomers. It seems plausible that a similar situation may be occurring in the salivary glands, where both PVF1 and PVF2 are needed in order to activate PVR and direct the salivary glands (Harris, 2007 and references therein).
Embryonic hemocytes can be visualized with an anti-Peroxidasin
antibody. Since no mutant was readily available
for the Pvr gene, the deletion strain
Df(2L)TE29Aa-14 was used to analyze any possible effect of a lack of
the gene. Hemocytes were observed in the mutant as well as in
the wild type, showing that Pvr is not essential for
hemocyte development. However, while the number was, at
the early developmental time points, about the
same as in the control, the distribution of the hemocytes
was hampered, especially on the dorsal side of the embryo in the deletion strain. At later stages, the antibody did not detect any hemocytes in the deficiency line. Since the deletion used uncovers ~25
genes predicted by the Genome Annotation Database of
Drosophila, the defective migration of hemocytes might
be due to deletion of other genes close to the Pvr
gene. However, other aspects of embryonic development
seem unaffected in the mutant embryos carrying
the deletion, i.e. segmentation and the positioning of the tracheal system, indicating that the phenotype observed cannot be an indirect consequence of
the disorganization of the embryonic development (Heino, 2001).
The border cells of the Drosophila ovary undergo a well-defined and developmentally regulated cell migration. Two signals control where and when the cells migrate. The steroid hormone ecdysone, acting through its receptor and a coactivator known as Taiman
, contributes to regulating the timing of border cell migration. PVF1, a growth factor related to platelet-derived growth factor and vascular-endothelial growth factor, contributes to guiding the border cells to the oocyte. To probe the mechanisms controlling border cell migration, a screen was performed for genes that exhibit dominant genetic interactions with taiman. Fourteen genomic regions were identified that interact with taiman. Within one region, Pvf1 was identified as the gene responsible for the interaction. Signaling by PVF1 has been proposed to guide the border cells to their proper target, but ectopic PVF1 has not been tested for its ability to redirect the border cells. The ability of PVF1 (as well as other factors such as Gurken) to guide the border cells to new targets was tested. Ectopic expression of PVF1 is sufficient to redirect border cells in some egg chambers but the other factors tested are not. These data suggest that the guidance of border cell migration is robust and that there are likely to be additional factors that contribute to long-range guidance of these cells. In addition, taiman and Pvf1 regulate the dynamic localization of E-cadherin in the border cells, possibly accounting for the interaction between these two pathways (McDonald, 2003).
The interaction of tai with Pvf1 appears to be specific because tai does not interact with either loss-of-function mutations or deficiencies that remove other genes known to regulate border cell migration, such as slbo or shotgun/DE-cadherin. Mutations in slbo or shotgun reduce DE-cadherin levels in the border cells, so tai does not interact with every gene that regulates DE-cadherin, possibly because tai regulates the distribution rather than the levels of DE-cadherin in the border cells. Identification of Pvf1 indicates that this screen provides a useful approach for identifying additional loci that affect border cell migration in general and regulate turnover of adhesion in particular (McDonald, 2003).
The genetic interaction between Pvf1 and tai indicates that the regulation of border cell migration timing and guidance might be linked. What is the nature of the interaction between tai and Pvf1 during border cell migration? Ecdysone signaling does not regulate PVF1 or PVR expression nor does Pvf1 regulate TAI expression, but the ecdysone and Pvf1 pathways both affect the distribution of DE-cadherin and Arm. A model is favored whereby tai and Pvf1 interact because they both regulate adhesion complex localization or turnover. The tai and Pvf1 genes could act independently to regulate cadherin-dynamics. Alternatively, tai and Pvf1 might function in a common pathway. TAI and PVR both function autonomously in the border cells, although they are unlikely to bind directly to each other because TAI localizes to the nucleus and PVR is a receptor tyrosine kinase localized to the membrane. One possibility is that PVR activates (or represses) the function of a protein whose expression is dependent on TAI, and that this protein in turn regulates cadherin dynamics in the border cells. Tyrosine phosphorylation of ß-catenin, the Arm homolog, causes destabilization of adhesion complexes in other cell types, so perhaps PVR activity destabilizes E-cadherin/Armadillo complexes specifically in the border cells. Identification of additional genes identified in this screen, in particular those that affect adhesion turnover in border cells, should help clarify the biochemical relationship between TAI and PVF1 (McDonald, 2003).
The results reported here demonstrate that ectopic expression of PVF1 is sufficient to redirect border cells even though, in Pvf1 null mutants, border cell clusters migrate normally in the majority of egg chambers. When PVF1 is ectopically expressed in random follicle cells, the border cells are attracted to these sources of PVF1. The border cells are attracted more efficiently to sources of PVF1 signal close to the anterior pole, indicating that they respond better to high concentrations of the ligand. The finding that doubling the dose of ectopically expressed PVF1 dramatically increases the frequency with which the cells respond to the ectopic signal confirms the idea of a concentration dependent effect (McDonald, 2003).
The concentration of ectopic PVF1 at the anterior end of the egg chamber appears to exceed the concentration of endogenous PVF1 at that position, even when only a single UAS-Pvf1 transgene is included in the experiment. Consistent with that idea, elimination of endogenous PVF does not significantly alter the response of the border cells to ectopic ligand. The border cells still migrate normally in many cases, apparently ignoring ectopically expressed PVF1. The most likely explanation for this is that there are additional germ-line-derived attractive cues that instruct the cells to migrate correctly in the absence of endogenous PVF1 and in the presence of ectopic PVF1 (McDonald, 2003).
PVF2 does not seem to be a good candidate for a redundant guidance cue because loss of function of the PVR receptor produces a phenotype that is indistinguishable from loss of PVF1 alone. Moreover UAS-Pvf2 was not able to redirect the border cells. This finding is surprising because PVF2 is thought to bind and activate the same receptor as PVF1. It is especially surprising because only PVF2 (expressed from the same UASPVF2 transgene) and not PVF1 is effective at misguiding hemocytes in the embryo. Together, these findings suggest a striking, and as-yet inexplicable, specificity of ligand action that will be interesting to study further (McDonald, 2003).
Gurken, the major Egfr ligand in the ovary, is not an effective guidance cue for the border cells, either when expressed alone or in combination with PVF1. The inability of Grk to affect border cells is striking because even class II and III phenotypes are absent, even though these are not uncommon following PVF misexpression. This is consistent with the observation that migration of the border cells to the oocyte is completely normal in grk mutant egg chambers and in mosaic egg chambers in which border cells lack EGF receptor function. Grk does, however, have a role in the dorsal migration of the border cells after they reach the oocyte. Currently, the evidence supporting a role for Grk in migration of the border cells to the oocyte is the combined effect of dominant-negative PVR and dominant-negative Egfr. Taken together with the results supplied in this study, the evidence in favor of a role for Egfr is somewhat better than the evidence in favor of a role for Grk, possibly suggesting the involvement of other Egfr ligands (McDonald, 2003 and references therein).
In addition to ligands for Pvr and Egfr, this study might imply the existence of other, as-yet-unidentified cues, that participate in the long-range guidance of the border cells. It is proposed that PVF1, and possibly additional unknown ligands, guide the border cells to the oocyte. Similarly, in the Drosophila central nervous system, multiple short-range and long-range cues are required to guide motor axons properly to their appropriate muscle targets. Perhaps even a simple migration, such as that of the border cells, uses multiple cues, each of which might only have a small contribution. Screens such as the one reported here might help identify the full set of border cell migration cues as well as additional genes that function in adhesion complex turnover (McDonald, 2003).
Cell death plays an essential role in development, and the removal of cell corpses presents an important challenge for the developing organism. Macrophages are largely responsible for the clearance of cell corpses in Drosophila melanogaster and mammalian systems. The developmental requirement for macrophages in Drosophila was examined and macrophage function was found to be essential for central nervous system (CNS) morphogenesis. Mutations were generated and analyzed in the Pvr locus, which encodes a receptor tyrosine kinase of the PDGF/VEGF family that is required for hemocyte migration. Loss of Pvr function causes the mispositioning of glia within the CNS and the disruption of the CNS axon scaffold. Inhibition of hemocyte development or of Croquemort, a receptor required for macrophage-mediated corpse engulfment, causes similar CNS defects. These data indicate that macrophage-mediated clearance of cell corpses is required for proper morphogenesis of the Drosophila CNS (Sears, 2003).
The Pvr locus was disrupted using homologous recombination. Ten independent homologous recombination events were recovered from the progeny of 3000 females, and the products of the targeting events were examined by a combination of long-range PCR and Southern blotting. Nine out of ten targeted insertions, including PvrKO2, yield the products expected for a successful targeting event in both PCR and Southern blot analysis. These alleles all failed to complement the lethality of Df(2L)TE128x11, a chromosomal deficiency that removes Pvr: the embryos complete embryogenesis but fail to hatch (Sears, 2003).
In addition to creating targeted insertions at the Pvr locus, multiple Pvr alleles were identified though a genetic non-complementation screen. Nine-thousand eight-hundred and ten lines of EMS-mutagenized flies were screened for failure to complement the lethality of Df(2L)TE128x11. One-hundred and eighty-five lethal or semi-lethal lines were recovered, of which 20 failed to complement PvrKO lethality. Among these putative Pvr alleles, four that were identified carry missense mutations in the extracellular domain, four that create stop codons in the extracellular domain and three that carry missense mutations in the kinase domain. The mutations in the kinase domain disrupt residues highly conserved among protein kinases. In Pvr729 an alanine residue in the catalytic loop, highly conserved among tyrosine kinases, is changed to a threonine. In Pvr4187 a glutamate residue in the activation loop, highly conserved among all protein kinases, is changed to a lysine. Finally, in Pvr7508 an aspartate residue in the DFG motif at the base of the activation loop is changed to a valine. Crystallographic data indicates this residue is involved in the divalent cation binding that contributes to nucleotide triphosphate recognition. The recovery of these changes in the kinase region are consistent with the importance of catalytic activity for Pvr function. All 11 of these EMS-induced Pvr alleles behave as embryonic lethal mutations (Sears, 2003).
Analysis was focused on three alleles predicted to yield severely truncated Pvr proteins. PvrKO2 contains two truncated copies of Pvr: one copy encodes a Pvr protein truncated after the fourth Ig domain; the other copy lacks promoter sequences and predicted start codons. Pvr9742 contains a stop codon immediately after the first Ig domain, while Pvr5363 has a 61-base deletion that removes amino acids 114-134 from the first Ig domain and replaces them with three new residues and a stop codon. Stage 16/17 PvrKO2, Pvr9742 and Pvr5363 embryos were examined for Pvr protein expression by Western blot using antisera raised against the C-terminal 275 amino acids of Pvr. Pvr expression was not detected in any of these mutant animals. PvrKO2, Pvr9742 and Pvr5363 give equivalent results in the studies described below (Sears, 2003).
CNS patterning was examined in Pvr mutants. CNS axons in the Drosophila embryo establish a precise pattern reiterated in each segment. CNS axons establish two longitudinal tracts that run the length of the embryo on either side of the midline, with a subset of these axons crossing the midline of the embryo, forming two commissural axon bundles per segment. CNS axons are guided in part by signals from glia precisely positioned at the midline and along the longitudinal tracts. Although CNS axon architecture is grossly normal in Pvr mutants, the precise ladder-like axon scaffold seen in wild-type embryos is disrupted. The scaffold in each segment has a rounded appearance, owing to changes in the separation between the anterior and posterior commissures and the longitudinal tracts (Sears, 2003).
As a metric of CNS shape change in Pvr mutants, the ratio of the ratio between the longitudinal tracts in each segment of late stage 16 embryos to the distance between anterior and posterior commissures was calculated. This ratio was significantly smaller in Pvr mutants than in wild-type or Pvr heterozygote controls (Sears, 2003).
Since glial cells within the CNS are required for proper axon tract formation, the positioning of glial cells was examined in Pvr mutants. Many CNS glial cells express the homeobox protein Repo and require Repo for their proper development. Repo-expressing glia form a patterned array along the longitudinal axon tracts and are largely excluded from the midline, except for a thin line of glia that enter the midline in each embryonic segment. In Pvr mutants, however, large numbers of Repo-positive glia accumulate in the midline. Thus, Pvr mutants have defects in both CNS axon tract morphology and glial positioning (Sears, 2003).
Since Pvr mutants show disruptions in CNS axon tract shape and glial cell positioning near the CNS midline, the pathfinding of CNS axons near the midline in Pvr mutants was examined in greater detail. The monoclonal antibody 1D4 (mAb 1D4) recognizes the Fasciclin 2 protein and labels a subset of longitudinal bundles that grow adjacent to the CNS midline. Mab1D4 is a commonly used tool for assessing axon fasciculation patterns and detecting inappropriate axon crossing of the CNS midline. Despite the changes in CNS axon scaffold shape and longitudinal glial distribution observed in Pvr mutants, no inappropriate axon crossing of the midline was detected. In addition, in wild-type animals three major tracts of Fasciclin 2-positive axons are observed near the dorsal surface of the CNS on either side of the midline. Three major tracts of Fasciclin 2-positive axons are also observed in Pvr mutants (Sears, 2003).
Although these tracts are relatively normal, they show very mild defasciculation in some segments, with axons in Pvr mutants occasionally separating from one another by greater distances than normal. Because disruptions in longitudinal glial cell development disrupt the formation of these axon bundles, the minor axon tract defects observed in Pvr mutants were not unexpected given the glial cell mispositioning seen in Pvr mutants (Sears, 2003).
To investigate the source of the CNS defects in Pvr mutants, the cell populations expressing Pvr were identified. Pvr protein is detected on several cell populations during embryonic development. In stage 16 embryos, Pvr is prominently expressed by cells at the surface of the embryo, as well as by cells scattered throughout the embryo and by cells at the CNS midline. The large number of Pvr-expressing cells scattered throughout the embryo are hemocytes, since they co-express the hemocyte marker Peroxidasin. The Pvr-expressing cells at the CNS midline are midline glia (a population distinct from the Repo-positive glia mentioned above) and are intimately associated with the CNS commissures. Pvr expression could not be detected in Pvr mutant embryos, confirming the specificity of the antiserum (Sears, 2003).
Drosophila midline glia play important roles in separating and wrapping CNS axon commissures. Although midline glia express Pvr and Pvr mutants exhibit defects in commissure morphology, no role for Pvr in the midline cells could be detected. To test directly whether Pvr acted in midline glia, high-level expression of a dominant-negative form of Pvr was driven in all midline cells using the Sim:Gal4 driver or specifically in midline glia using the Slit:Gal4 driver. However, in neither case was a detectable CNS axon or Repo-positive glia phenotype generated (Sears, 2003).
Is the CNS phenotype related to Pvr expression in hemocytes? In Pvr mutants, hemocytes largely fail to migrate away from their birthplace in the head. To test whether Pvr function in hemocytes is important for CNS development, expression of dominant-negative Pvr was driven in the developing hemocytes. While no solely hemocyte-specific Gal4 driver is available, Gcm:Gal4 can be used to drive gene expression in embryonic hemocytes. Gcm:Gal4 drives gene expression specifically in hemocytes beginning at stage 11 and later, beginning at stage 15, in other cells that do not detectably express Pvr. Embryos expressing dominant-negative Pvr under the control of Gcm:Gal4 have hemocyte migration defects resembling those of Pvr mutants, consistent with Pvr acting cell-autonomously to control hemocyte migration. Most importantly, embryos expressing dominant-negative Pvr under Gcm:Gal4 control also exhibit rounding of CNS axon commissures and mispositioning of Repo-positive glial cells similar to Pvr mutants. These data are consistent with Pvr acting in hemocytes to control CNS patterning and suggest that hemocyte function is required during CNS development (Sears, 2003).
To further examine the potential contribution of hemocytes to CNS development, animals mutant for serpent (srp), which encodes a GATA-family transcription factor required for hemocyte development, were examined. srpneo45 is a hemocyte-specific allele of serpent, and srpneo45 animals lack all hemocytes. Examination of srpneo45 embryos demonstrated that not only do srpneo45 mutants lack macrophages, they also exhibit CNS axon scaffold defects similar to those in Pvr mutants, with characteristic rounding of commissures. Quantitative representation of CNS axon tract morphology in srpneo45 mutants confirmed this observation. srpneo45 animals also show longitudinal glia positioning defects similar to those seen in Pvr mutants. Thus, mutants that disrupt either hemocyte production or migration cause similar alterations in CNS morphogenesis (Sears, 2003).
One possible explanation for dependence of CNS morphogenesis on hemocytes is that hemocyte-derived macrophages are needed to engulf cell corpses generated during development. To test this possibility animals in which macrophages appear to develop normally, but fail to engulf cell corpses, were examined. This was achieved using animals with reduced function of crq, which encodes a CD36-related receptor required for Drosophila macrophages to engulf dead cells. crq loss-of-function was examined using RNAi. Embryos injected with dsRNA corresponding to either of two non-overlapping regions within the Crq transcript had CNS axon scaffold defects similar to those in Pvr and srp mutants. In crq RNAi embryos the ratio of distance between longitudinals to distance between commissures was significantly different from wild type, but not significantly different from Pvr or srp mutants. In addition, crq RNAi animals also showed defects in the positioning of Repo-positive glia similar to those seen in Pvr and srp mutants. These data further support the importance of hemocytes in CNS morphogenesis and specifically suggest that engulfment of dead cells by hemocyte-derived macrophages is essential for CNS development (Sears, 2003).
The Drosophila PDGF/VEGF receptor (PVR) has known functions in the guidance of cell migration. It has been demonstrated that during embryonic hematopoiesis, PVR has a role in the control of antiapoptotic cell survival. In Pvr mutants, a large fraction of the embryonic hemocyte population undergoes apoptosis, and the remaining blood cells cannibalistically phagocytose their dying peers. Consequently, total hemocyte numbers drop dramatically during embryogenesis, and large aggregates of engorged macrophages form carrying multiple apoptotic corpses. Hemocyte-specific expression of the pan-caspase inhibitor p35 in Pvr mutants eliminates hemocyte aggregates and restores blood cell counts and morphology. Additional rescue experiments suggest involvement of the Ras pathway in PVR-mediated blood cell survival. In cell culture, PVR has been demonstrated to directly control survival of a hemocyte cell line. This function of PVR shows striking conservation with mammalian hematopoiesis and establishes Drosophila as a model to study hematopoietic cell survival in development and disease (Brückner, 2004).
These results demonstrate that PVR controls the trophic survival of embryonic blood cells in vivo and an embryonic hemocyte cell line in vitro. Inhibition of PVR signaling or loss of Pvr function results in caspase-dependent apoptotic hemocyte death and formation of large blood cell aggregates. Blood cell clustering in Pvr mutant embryos has previously been interpreted as a migration defect. Based on the current findings, a different scenario is proposed: Dying hemocytes lose their ability to migrate, which may happen in many cases before cells leave the anterior locations of their origin. The resulting apoptotic corpses pose a strong attractant for their still-viable peer macrophages, which migrate toward them to fulfil their phagocytic function. Subsequently, cannibalistic phagocytosis leads to engorgement of macrophages with multiple blood cell corpses and results in the overall appearance of large hemocyte aggregates (Brückner, 2004).
Blood cell death in Pvr mutants occurs progressively throughout embryonic development. While hemocyte clustering in Pvr1 and Pvr4 becomes evident at different stages of embryonic development, blood cell aggregates persist until the end of embryogenesis and intensify over time. This is consistent with observations in the hemocyte cell line Kc 167, where silencing of Pvr results in apoptotic cell death over the course of days. Slow hemocyte death suggests activity of other partially redundant signaling pathways supporting cell survival. The Jak/Stat and Toll/Cactus pathways have been proposed to play roles in larval hemocyte survival, and it remains to be shown whether these pathways have a trophic function in the embryonic hematopoietic system. Further, it needs to be determined whether Pvr mutant blood cells die in a stochastic manner, or whether macrophages comprise different sublineages with variable dependence on trophic PVR signaling. While these studies indicate that both dying and actively phagocytosing blood cells express the same macrophage markers, identification of additional markers may reveal a distinction between sublineages (Brückner, 2004).
Defects in hemocyte function may indirectly account for the associated lethality of Pvr mutants. Pvr mutants suffer from secondary defects in the morphogenesis of the embryonic central nervous system. These defects are caused by a failure of hemocytes to remove dead cells; similar effects were seen in embryos that lack srp function, or in which phagocytosis is impaired. It is likely that additional secondary defects of Pvr mutants will be found in other organ systems that rely on reshaping by the phagocytic activity of macrophages (Brückner, 2004).
Trophic survival is well known in mammalian systems, where it serves as an important mechanism to control cell number -- extracellular factors produced by target tissues trigger survival pathways that suppress apoptotic cell death and thereby control cell number. In Drosophila, few rare described cases of antiapoptotic survival signaling are mediated through activation of the Drosophila EGFR (DER) by its ligands Vein or Spitz in the survival of glia cells, and by both ligands in the survival of smooth cuticle cells. Other signaling pathways may have similar roles during development, but detailed analysis is often limited. In accordance with the classical model of trophic cues, the PVR ligands PVF1-3 are expressed dynamically in multiple embryonic tissues throughout embryogenesis. Only simultaneous RNAi silencing of all three ligands leads to hemocyte aggregation, consistent with PVF1-3 providing the crucial stimuli for hemocyte survival by activation of PVR (Brückner, 2004).
Pvr mutant rescue experiments demonstrate that activated Ras is sufficient to restore hemocyte survival. This result resembles findings from survival signaling by DER, which was shown to inhibit action of the proapoptotic protein HID by phosphorylation through Ras-activated MAPK. Since PVR signaling triggers MAPK activation in Schneider cells and may have the same effect in embryonic hemocytes, it is likely that the Ras/MAPK pathway is a route of antiapoptotic PVR signaling. Expression of dominant-negative RasN17 did not lead to large hemocyte aggregates but induced mild enlargement of hemocytes at a low penetrance. This mild phenotype points to weak defects in blood cell survival. The incomplete effect of RasN17 may be due to a number of reasons. RasN17may be too weak to fully block endogenous Ras signaling, or Ras signaling may be redundant with other signaling pathways that are active in PVR-dependent cell survival. In Pvr1 rescue experiments, activated RasV12 was used, which was shown to ectopically activate other signaling pathways such as the PI3K pathway. Therefore, rescue by RasV12 may involve a number of downstream pathways, but the Ras/MAPK pathway itself may still be central to the observed effect, consistent with a current model for apoptosis in Drosophila. Regardless of the upstream pathways involved, inhibition of caspases by the baculovirus inhibitor of apoptosis p35 was sufficient to rescue the Pvr mutant hemocyte death and aggregation phenotype. In these rescue experiments, p35 appeared slightly less potent than RasV12, which may be due to the inability of p35 to inhibit the upstream caspase Dronc, or partially insufficient expression levels, since p35 inhibits caspases by stoichiometric binding (Brückner, 2004).
While activated lambdaPVR and other activated rescue transgenes efficiently revert hemocyte clustering and cell death, embryos still retain residual defects in hemocyte distribution. In stage 11/12 embryos, blood cell entry into the posterior end of the elongated germ band is affected, and in stage 15/16 embryos, the posterior area of the ventral nervous system is lacking hemocytes. This is consistent with a
role for PVR in the entry/migration of hemocytes into the posterior end of stage 11/12 embryos. Since the natural route of hemocytes is to distribute from the posterior end into ventral posterior areas, it is proposed that the observed ventral-posterior lack in hemocytes may reflect the initial inability of blood cells to invade, or migrate within, the posterior end. Alternatively, PVR activity may be required at multiple steps of embryonic hemocyte migration. In this case, defects at early steps of hemocyte migration (i.e., entry into the posterior end) may be more readily visualized than defects at later steps, which may be masked by additional independent mechanisms of hemocyte dispersal. To distinguish between these possibilities, more refined techniques to study blood cell migration will be required. Consistent with both models, uniform expression of activated lambdaPVR in hemocytes leads to a similar, yet milder, phenotype at a low penetrance, suggesting that correct hemocyte entry or migration requires tightly controlled or locally asymmetric activation of PVR. By analogy, migration of border cells in the egg chamber is dramatically affected by uniform activation of PVR, revealing a role for locally asymmetric PVR activation in the guidance of border cells (Brückner, 2004).
In mammals, PDGF/VEGF receptors are known to mediate additional cellular responses, such as cell proliferation and differentiation. Drosophila PVF2 has been described to promote proliferation of larval hemocytes. The current experiments do not point to a role for PVR in the proliferation of embryonic blood cells, since Pvr RNAi-treated Kc cells did not show an obvious arrest in the cell cycle, and p35 was sufficient to rescue blood cell counts in mutant embryos. Since blood cell proliferation occurs very early during embryonic development, it cannot be excluded that an unnoticed maternal contribution of Pvr may obscure a role for the receptor in embryonic hemocyte proliferation in vivo. Likewise, with respect to blood cell differentiation, no effects of Pvr loss-of-function on macrophage differentiation were detected as judged by the marker Crq. Nevertheless, a role for PVR in hemocyte differentiation may become apparent once more specific lineage markers become available (Brückner, 2004).
Taken together, PVR has at least two functions in the embryonic hematopoietic system: (1) PVR mediates antiapoptotic survival of blood cells throughout embryonic development, and (2) PVR is required for invasion into/migration within the posterior end of the embryo. Rescue of cell viability reverts the dramatic blood cell aggregation phenotype and leads to dispersal of cells in anterior and dorsal areas of the embryo (Brückner, 2004).
The role of Drosophila PVR in trophic cell survival emphasizes the high degree of conservation between Drosophila and vertebrate PDGF/VEGF family receptor function. In vivo and cell culture work now provides the basis to study cell survival in a simple but highly conserved hematopoietic system. In vertebrates, control of cell survival is an important aspect of hematopoiesis and stem cell maintenance. Antiapoptotic cell survival and its aberrant prolongation are a major mechanism in the formation of human neoplasias. In many cases, connections to deregulated upstream signaling pathways remain unclear. Interestingly, in Acute Myeloid Leukemias (AML), more than one-third of cases are associated with specific activating mutations in the PDGF/VEGF receptors Flt3 and c-Kit, and activating fusions of PDGFßR replace the more common oncogenic BCR-ABL fusions in some cases of Chronic Myeloid Leukemia (CML). The contribution of these and other disease-associated genes to aspects of cell survival versus proliferation is still difficult to assess in vivo, yet their mechanism of action is important for the selection of molecularly targeted therapies. Drosophila embryonic hematopoiesis allows the in vivo study of blood cell survival independent of cell proliferation, and this work has demonstrated the antiapoptotic potential of the Drosophila PDGF/VEGF receptor and activated signaling components such as RasV12. It will be interesting to exploit the system further by testing disease-related genes of the same and particularly other families for their in vivo potential to rescue blood cell survival in Drosophila. Complementary to these in vivo findings, a Drosophila cell culture system was established for the study of PVR-dependent blood cell survival. Genome-wide RNAi screens will allow identification of modifiers of PVR-dependent blood cell survival (Brückner, 2004).
Condensation is a process whereby a tissue undergoes a coordinated decrease in size and increase in cellular density during development. Although it occurs in many developmental contexts, the mechanisms underlying this process are largely unknown. This study investigated condensation in the embryonic Drosophila ventral nerve cord (VNC). Two major events coincide with condensation during embryogenesis: the deposition of extracellular matrix by hemocytes, and the onset of central nervous system activity. Preventing hemocyte migration by removing the function of the Drosophila VEGF receptor homologue, Pvr, or by disrupting Rac1 function in these cells, inhibits condensation. In the absence of hemocytes migrating adjacent to the developing VNC, the extracellular matrix components Collagen IV, Viking and Peroxidasin are not deposited around this tissue. Blocking neural activity by targeted expression of tetanus toxin light chain or an inwardly rectifying potassium channel also inhibits condensation. Disrupting Rac1 function in either glia or neurons, including those located in the nerve cord, causes a similar phenotype. These data suggest that condensation of the VNC during Drosophila embryogenesis depends on both hemocyte-deposited extracellular matrix and neural activity, and suggest a mechanism whereby these processes work together to shape the developing central nervous system (Olofsson, 2005).
Thus, disrupting hemocyte migration inhibits VNC condensation in the embryo. Lack of hemocyte migration is associated with a severe reduction of ECM components (Collagen IV and Peroxidasin) throughout the embryo and more particularly a loss of these components around the VNC. This leads to a proposal that correct assembly of the ECM depends on hemocytes, and that basement membrane is required for condensation. Supporting a role for ECM in VNC condensation, defects are observed in loss-of-function mutants of integrins, which are ECM receptors and appear themselves to be required for correct assembly of basement membranes. Mutants in integrins or the ECM component Laminin A share at least one other phenotype with embryos in which hemocyte migration has been inhibited: gut morphogenesis is impaired. Thus, a dysfunctional ECM may explain several of the morphogenetic defects seen in embryos with defective hemocyte migration (Olofsson, 2005).
How might basement membrane contribute to VNC condensation? Basement membrane may serve as a substrate for cellular movements involved in condensation and/or regulate signaling events relevant to condensation. Basement membrane is also required for normal neuromuscular junction development, and might be part of the functional blood-brain barrier in Drosophila. Hence, neural function may be disrupted when basement membrane formation is inhibited. However, condensation phenotypes in embryos with impeded hemocyte migration are more severe than in embryos in which neural activity has been blocked. This argues that the condensation phenotype seen in hemocyte migration-blocked embryos cannot be explained simply by a loss of neural activity (Olofsson, 2005).
Although animals in which hemocyte migration is blocked fail to deposit Collagen IV appropriately, it has not been demonstrated that Collagen IV function is required for condensation. However, embryos expressing a dominant negative form of Collagen IV under the control of a heatshock promoter fail to condense their nerve cord. While these data point towards a functional role of Collagen IV in condensation, further studies will be necessary to clarify the specific role of Collagen IV during condensation (Olofsson, 2005).
This study has not investigated whether phagocytosis of cells within the VNC contributes to condensation. pvr mutants show a perdurance of unengulfed cells at the ventral surface of the CNS at stage 14. The majority of these cells seem to disappear later, possibly engulfed by epidermal cells. pvr mutants also maintain some very restricted points of attachment between the epidermis and the VNC. This phenotype is not observed when hemocyte migration is blocked using mutant Rac1 expressed by crq-GAL4. This likely reflects failure of hemocyte migration at a later stage, after the two tissues have separated (Olofsson, 2005).
The major cell type that engulfs apoptotic corpses within the CNS is the subperineural glia. In the absence of macrophages (in the Bic-D mutants), apoptotic cells are still expelled from the CNS but accumulate at the ventral surface, similar to the observations in the pvr mutant. Hemocytes are required for normal CNS morphogenesis: at stage 16, pvr mutants and Crq RNAi treated embryos have mispositioned glia and minor axon scaffolding defects. These data were interpreted to reflect a failure of engulfment of cell corpses. In the context of these findings, an additional cause for glial mispositioning in pvr mutant embryos could be a loss of basement membrane components and the failure to condense (Olofsson, 2005).
VNC condensation correlates with the onset of neural activity in the CNS, and it is found that expressing tetanus toxin light chain or the inwardly rectifying K+ channel Kir2.1 pan-neuronally impairs condensation. This suggests that neural activity influences normal condensation. Neural activity could contribute to condensation in multiple ways. It could directly regulate cellular events relevant to condensation, such as adhesion or actin-based motility, or activity could influence the transcription of genes relevant to such events. Alternatively, neural activity could maintain synaptic connectivity among cells necessary for condensation, rather than directing changes in cellular behavior leading to condensation. Some condensation occurs before neural activity begins, and the condensation phenotypes resulting from impeding hemocyte migration are more severe than those resulting from blocking neural activity. This suggests that there may be multiple stages of condensation, including an earlier activity-independent stage and a later stage that is influenced by activity (Olofsson, 2005).
VNC condensation can be inhibited by expressing mutant Rac1 in lateral glia or
neurons. In glia, migration and ensheathing behaviors require cytoskeletal integrity. When mutant Rac1 is expressed in peripheral glia, the formation of cellular extensions is disrupted, and this is accompanied by glia migration and axon ensheathment defects. Similarly, ensheathment of longitudinal axon tracts by longitudinal glia is disrupted in htl loss of function embryos. The VNC condensation phenotype in these embryos is interpreted as indication that glia need dynamic actin cytoskeleton to generate a condensing force. Two types of VNC glia are particularly well placed to generate such a force: longitudinal glia associated with VNC longitudinal connectives, and perineural glia, which ensheath the cortex of the VNC. Cell-cell contacts and cell-ECM contacts among these cells accompanied by remodeling of extracellular matrix could help generate a condensing force within and across neuromeres through changes in cell shape, adhesion or migration. A similar process occurs during mesenchymal condensation (Olofsson, 2005).
In neurons, neurite extension requires normal Rac GTPase activity. Expressing mutant Rac1 in these cells causes defects in axonal outgrowth. In wild type animals, VNC axons are arranged into longitudinal connectives that extend along the length of the nerve cord, and these are well placed to generate an anteroposterior condensing force. This could happen through differential cell adhesion of neurites within the longitudinal connectives or overall shortening of the axons. The observation that axons in VNC longitudinal connectives loop out during condensation in metamorphic insects is consistent with this idea. It is interesting to note that condensation is inhibited in embryos in which mutant Rac1 is expressed in glia, but longitudinal axon tracts appear normal in these animals. This suggests that if axons help generate a condensing force, they likely do this with the help of glia, possibly using these cells as a substrate (Olofsson, 2005).
It is also possible that at least part of the force required for condensation may come from outside the VNC. Somatic muscles connect to the VNC during embryogenesis, and embryonic muscle activity toward the end of embryogenesis is well timed for generating such a force. Also, the methods used to manipulate glia or neuron development in this study may affect neuromuscular activity by disrupting blood-brain barrier formation, or by affecting the Rac-dependent formation of synaptic structures. However, the observation that the CNS can condense in mutants in which muscles do not form normally argues against a major contribution from muscle activity (Olofsson, 2005).
These data identify several areas for further investigation. By following the behavior of small populations of cells in the VNC it may possible to analyze in vivo changes associated with the condensation process and get insight into how changes in organ shape are generated and coordinated. It will also be interesting to examine the contributions made by components of the ECM to normal blood-brain barrier function. Finally, it may be possible to use VNC condensation in embryonic Drosophila to investigate the molecular and cellular basis of how neural activity is translated into a morphogenetic event (Olofsson, 2005).
Drosophila hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. The migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and Pvf3; these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, PI3K is shown to be essential for hemocyte chemotaxis toward wounds and Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. These results demonstrate that at least two separate mechanisms operate in Drosophila embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis (Wood, 2007).
During Drosophila embryogenesis, hemocytes derive exclusively from head mesoderm at around 2 h after gastrulation. From this point of origin, these cells migrate along stereotypical routes to populate the whole embryo by stage 17. It has been shown that the developmental migration of these cells is dependent on the expression of the VEGF/PDGF ligands Pvf1, Pvf2, and Pvf3. The PDGF/VEGF receptor (PVR) is expressed in hemocytes, and pvr mutant embryos fail to exhibit normal hemocyte migrations, resulting in an accumulation of these cells at their head end. A recent study has demonstrated a role of PVR in controlling anti-apoptotic cell survival of embryonic hemocytes and suggests that the defect in hemocyte distribution observed in the mutant is largely due to high numbers of hemocytes undergoing apoptosis and becoming engulfed by their neighbors. However, this study also showed that Pvr expression within hemocytes is required for the directed migration of a subset of these cells that enter the extended germ during normal development, suggesting that this population of hemocytes may well be using Pvf signals as a chemoattractant to guide their migrations. Additionally, ectopic expression of Pvf2 within the embryo has been shown to be sufficient to induce a chemotactic response from embryonic hemocytes (Wood, 2007).
In addition to migrating along developmental pathways, embryonic hemocytes have been shown to migrate toward a laser-induced wound in a process that resembles the vertebrate inflammatory response. For a hemocyte to chemotax toward a chemotactic source, be it a wound or a guidance cue expressed along developmental migration routes, it has to be able to sense a chemotactic gradient and polarize in alignment with that gradient. Studies using Dictyostelium discoideum and mammalian neutrophils have demonstrated that the phosphoinositides PtdIns(3,4,5)P3 (PIP3) and PtdIns(3,4)P2 (PIP2) are key signaling molecules that become rapidly and highly polarized in cells that are exposed to a gradient of chemoattractant. In these actively chemotaxing cells, phosphoinositide 3-kinases (PI3Ks) rapidly translocate from the cytosol to the membrane at the leading edge of the cell, whereas phosphatase and tensin homologue (PTEN) dissociates from the leading edge and becomes restricted to the sides and the rear. The difference in localization of these two enzymes leads to localized PIP3 production at the leading edge of the cell. Down- or up-regulation of PIP3 by deletion of PI3Ks or of PTEN, respectively, results in severely reduced efficiency of chemotaxis. Though PI3K has been shown to be important for cell motility using these model systems, its role for single-cell chemotaxis in vivo in a multicellular organism has yet to be clarified. D. melanogaster has one class I PI3K, Dp110, whose role in cell growth control and cell survival has been well characterized; however, no role in cell migration and chemotaxis in Drosophila for this protein has been shown (Wood, 2007).
This study analyzed the developmental migrations of hemocytes and characterized in detail their migration patterns along the ventral midline. Quantitative analysis shows that ventral midline hemocytes undergo a rapid lateral migration, during which they are highly polarized. Pvf2 and -3 expression in the central nervous system (CNS), and Pvf2 alone in the dorsal vessel, are essential for directing the migration of hemocytes along these structures, and a decrease in expression of these ligands in the CNS is essential for the normal lateral migration of hemocytes in this region. The function of PI3K was analyzed in hemocytes. Using both dominant-negative PI3K-expressing hemocytes and the specific PI3K inhibitory drug LY294002, PI3K is shown not to be required for the Pvf-dependent normal dispersal of hemocytes during development but is essential for chemotaxis toward wounds. Additionally, hemocyte chemotaxis toward wounds is shown to be dependent on actin polymerization but that PI3K is not required for lamellipodial formation and instead appears to be required to sense a chemotactic gradient from a wound and polarize the hemocyte accordingly. These results demonstrate that at least two separate mechanisms operate in Drosophila embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the Pvf growth factor signals that coordinate their developmental migrations along the ventral midline and dorsal vessel during embryogenesis (Wood, 2007).
Many obvious parallels exist between the migration of hemocytes along the ventral midline CNS and another developmentally regulated migration in Drosphila, that of border cell migration. Border cells take ~6 h to migrate a distance of 100 µm, a speed consistent with that describe for hemocyte migration along the CNS. Successful border cell migration, like hemocyte migration, requires the expression of the Pvr in the migrating cells and, just as was seen for hemocytes, the chemotactic signals detected by the PVR in the border cells are not transduced through PI3K. Successful migration of border cells does, however, require Rac signaling and the Rac activator myoblast city (mbc), the D. melanogaster homologue of Dock 180. It has been shown that hemocyte-specific expression of dominant-negative RacN17 disrupts all hemocyte developmental migrations, demonstrating that Rac is required for the successful migration of ventral midline hemocytes along the CNS. Given that Pvr couples to the Dock 180 signaling pathway during border cell migration and that Dock 180 has been shown to be involved in the migration of lymphocytes, Mbc/Dock 180 is a potentially important protein for hemocyte migration. Despite the fact that mbc mutant embryos display a grossly normal pattern of hemocyte dispersal, it would be interesting to look in detail at the migration of these mutant cells along the ventral nerve cord. More work is needed to investigate what other similarities may exist between border cell migration and ventral midline hemocyte migration. During development, only a subset of the hemocytes present in the embryo respond to the midline Pvf expression and migrate along the CNS accordingly. Other cells follow other migratory pathways. What specifies these cells to migrate along the midline? Important studies in border cell migration have shown that the JAK-Stat signaling pathway signaling through the Domeless receptor (Dome) is necessary and sufficient to transform nonmotile epithelial cells into invasive ones. Whether a similar signaling mechanism is operating to specify future ventral midline hemoctyes and initiate their migration remains to be seen (Wood, 2007).
From stage 14 onwards, once hemocytes occupy the entire ventral midline, individual cells begin to rapidly leave the midline and occupy more lateral positions. At this stage of development, hemocytes appear to be highly polarized, exhibiting large lamellipodia at their leading edges and migrating at a speed more than three times faster than their earlier midline migration. This lateral movement requires a down-regulation in the attractive signal provided by Pvf2 in the midline, but is this the only driving force for the lateral movement? One possibility is that a different source of chemoattractant exists in the more lateral positions and that once Pvf2 expression is sufficiently down-regulated, this chemoattractant source operates to pull hemocytes laterally. Alternatively, hemocytes may be actively repelled from the midline or from one another, and the lateral migration observed by a subset of these hemocytes is a consequence of these cells attempting to maximize the distance between one another while maintaining contact with the CNS. It remains to be seen which, if any, of these hypotheses is true, but what is certain is that the guidance of hemocytes along the ventral midline of the embryo is not as simple as was first thought, and more studies are required to determine the exact relationship between this subpopulation of hemocytes and the different structures within the CNS as well as the overlying ectodermal cells, any of which could provide either chemoattractants or repellents for the migrating hemocytes to respond to (Wood, 2007).
This study has demonstrated a requirement of PI3K for the polarization and active chemotaxis of hemocytes toward an epithelial wound. This is the first demonstration of the role of PI3K for single-cell chemotaxis in Drosophila and shows a striking correlation with the mechanism of cell chemotaxis used by D. discoideum and mammalian neutrophils. In these model systems, class I PI3Ks are activated upon stimulation of G protein-coupled chemoattractant receptors and, once activated, PI3Ks catalyze the production of the phosphoinositides PIP3 and PIP2 at the leading edge of the cell. The accumulation of PIP3/PIP2 leads to a rapid and transient recruitment of pleckstrin homology domain-containing proteins, including the serine/threonine kinase Akt/PKB. Akt/PKB itself becomes activated upon recruitment to the membrane and, in D. discoideum, activates the serine/threonine kinase p21-activated kinase a, which eventually leads to the phosphorylation of Myosin II and subsequent polarization of the cytoskeleton. Evidence also exists to support a role for the PI3K antagonist PTEN in helping to establish and maintain the intercellular PIP3 gradient required for successful chemotaxis by down-regulating the PIP3 pathway at the rear of the migrating cell. How much of this signaling pathway is operating in chemotaxing hemocytes remains to be seen. The current study demonstrates the involvement of PI3K, and previous work has shown that the small GTPase Rac is required for efficient hemocyte chemotaxis toward wounds. In neutrophils, PIP3 production has been shown to be autocatalytic and to require Rac but not Cdc42. In the proposed positive feedback loop, it is thought that PIP3 may stimulate Rac through activation of a specific Rac GEF, which in turn activates PI3K, as well as effectors that mediate lamellipodial protrusion. Because Rac is absolutely required for hemocyte chemotaxis and lamellipodia formation, it is tempting to speculate that a similar feedback loop may be operating in Drosophila hemocytes. Further work is required to determine the complex relationships operating among PI3K, Rho family small GTPases, and the actin cytoskeleton that coordinate chemotactic migration in these highly motile cells (Wood, 2007).
The PI3K-dependent mechanism of polarization required for hemocyte chemotaxis toward a wound is extremely fast and perfectly suited for mature, highly motile hemocytes that need to rapidly react to a source of attractive signal, be it a wound, an invading organism, or an apoptotic cell. In contrast, the mechanics to developmentally disperse need not be so rapid, since the aim during development is simply to ensure that hemocytes migrate toward and arrive at their target tissue in a given amount of time and does not require the rapid response to constantly changing environments required for mature hemocytes. The mechanism controlling the developmental migration of hemocytes along the ventral midline is consequently much slower and is dependent on slow-diffusing growth factors of the Pvf family providing short-range guidance information signaling through the receptor tyrosine kinase PVR. These two mechanisms may not be the only ways in which hemocytes are able to chemotax toward an attractive source; indeed, the observation that hemocytes travel different migratory routes in the embryo suggests that they may not all be using the same machinery to polarize and migrate. What does seem to be consistent for both chemotaxis toward developmental signals and toward wounds, like motility in many cell types, is a requirement for Rac signaling and the formation of actin protrusions (Wood, 2007).
The fact that hemocyte migrations within the embryo are strictly regulated and adhere to a stereotyped pattern is important in a developmental context. Throughout embryogenesis, hemocytes carry out important developmental functions within the embryo, such as the engulfment and removal of apoptotic cells and the laying down of many extracellular matrix molecules, including collagen IV and laminin, that compose the basement membrane surrounding internal organs. The failure of hemocytes to travel along their normal migratory routes therefore has serious consequences. Such defects have been described in pvr mutants, where a lack of hemocyte migration along the ventral nerve cord results in a failure in CNS condensation, as well as a disruption in axon patterning. It is therefore vital for the embryo to ensure that hemocytes arrive at their correct target tissues during development. For this to occur, it is not sufficient to allow these cells to passively disperse throughout the embryo by random migrations; instead, a directed and tightly controlled migration is required (Wood, 2007).
In this study, drugs were directly applied to Drosophila embryos using bead implantation. The application of drugs has been a powerful tool in cell culture and in vitro cell motility studies but remains largely unused in Drosophila. Using a bead assay, it will be possible to take advantage of the many useful drugs available to block both specific signaling pathways as well as important cytoskeletal processes. Combined with the powerful genetics available in Drosphila and the relative ease of live imaging in this system, the study of Drosophila hemocytes provides a powerful model to address the process of cell motility and chemotaxis and will undoubtedly provide a clearer understanding of the regulation and mechanics of single-cell migration in the complex setting of a multicellular organism (Wood, 2007).
Although directed migration is a feature of both individual cells and cell groups, guided migration has been studied most extensively for single cells in simple environments. Collective guidance of cell groups remains poorly understood, despite its relevance for development and metastasis. Neural crest cells and neuronal precursors migrate as loosely organized streams of individual cells, whereas cells of the fish lateral line, Drosophila tracheal tubes and border-cell clusters migrate as more coherent groups. This study used Drosophila border cells to examine how collective guidance is performed. It is reported that border cells migrate in two phases using distinct mechanisms. Genetic analysis combined with live imaging shows that polarized cell behaviour is critical for the initial phase of migration, whereas dynamic collective behaviour dominates later. PDGF- and VEGF-related receptor and epidermal growth factor receptor act in both phases, but use different effector pathways in each. The myoblast city (Mbc, also known as DOCK180) and engulfment and cell motility (ELMO, also known as Ced-12) pathway is required for the early phase, in which guidance depends on subcellular localization of signalling within a leading cell. During the later phase, mitogen-activated protein kinase and phospholipase Cγ are used redundantly, and it was found that the cluster makes use of the difference in signal levels between cells to guide migration. Thus, information processing at the multicellular level is used to guide collective behaviour of a cell group (Bianco, 2007).
Border cells perform a well-defined, invasive and directional migration during Drosophila oogenesis. They delaminate from the follicular epithelium at the anterior end of an egg chamber and migrate posteriorly, towards the oocyte, as a compact cluster. They then migrate dorsally towards the oocyte nucleus. The border-cell cluster consists of about six outer migratory border cells and two inner polar cells that induce migratory behaviour in the outer cells but seem to be non-migratory. Two receptor tyrosine kinases (RTKs), PDGF- and VEGF-related receptor (PVR) and epidermal growth factor receptor (EGFR), are guidance receptors for border cells. Both receptors act redundantly during posterior migration towards the oocyte, whereas EGFR and its dorsally localized ligand, Gurken, are essential for dorsal migration. Localized signalling from the RTKs is important and actively maintained, especially early in migration. Rac and the atypical Rac exchange factor Mbc (myoblast city, also known as DOCK180) are important effectors. To determine the contribution of Mbc and related proteins, a loss-of-function allele of their common cofactor ELMO (engulfment and cell motility, also known as Ced-12) was generated by homologous recombination. Clusters of elmo mutant border cells arrested early in migration, a defect that could be rescued by expressing elmo complementary DNA. As for mbc, reduction in elmo function suppressed F-actin accumulation caused by constitutive PVR signalling, placing ELMO downstream of the receptor in this respect (Bianco, 2007).
To determine whether later steps in migration also depend on ELMO, mosaic border-cell clusters consisting of wild-type and mutant cells were investigated. If a mutation does not affect migration, mutant cells should be distributed randomly within the cluster. Mutant cells defective in migration would be in the rear, 'carried along' by normal cells. As expected, Pvr and Egfr double mutant cells were in the rear during posterior migration, as were Egfr mutant cells during dorsal migration, reflecting the requirements at each stage. elmo mutant cells were in the rear during the initial migration, but were equally frequent in the leading position during dorsal migration. This indicates that, although ELMO is essential for the early-phase signalling, the later phase of migration does not require the Mbc-ELMO complex (Bianco, 2007).
To understand late guidance signalling, EGFR signalling, on which dorsal migration depends, was dissected. Uniformly activated EGFR, like PVR, dominantly impairs migration. The carboxy-terminal tail of EGFR was essential for this activity. Systematic mutagenesis of all docking tyrosines to phenylalanine identified Y1357 as being critical, with minor contributions from Y1405 and Y1406. Other tyrosines, including Y1095 in the conserved activation loop (phosphorylated in HER2 (Human EGF Receptor 2), were not required. Twenty Src-homology 2- and phosphotyrosine-binding-containing signalling molecules were tested for binding to active EGFR and tyrosine mutants. Y1357 was necessary and sufficient for binding of the adaptor protein Shc and its phosphotyrosine-binding domain. No other tested interactor behaved in this way. Binding was confirmed by immunoprecipitation. Border cells mutant for Shc showed no dorsal migration and, when PVR signalling was also blocked, these cells showed severely impaired posterior migration. This phenotype is identical to that of Egfr mutant cells, suggesting that Shc is essential immediately downstream of EGFR for guidance signalling (Bianco, 2007).
The Shc adaptor protein links EGFR and other RTKs to mitogen-activated protein kinase (MAPK) kinase signalling as well as to other classical downstream pathways. Raf, phospholipase Cγ (PLC-γ) or phosphatidylinositol-3-OH-kinase are not uniquely required for migration; however, the pathways might act redundantly. Simultaneous perturbation of PLC-γ and Raf impaired migration, with no effect of phosphatidylinositol-3-OH kinase. Double mutant border-cell clusters, cell-autonomously lacking PLC-γ and Raf or lacking PLC-γ and MAPK kinase (MAPKK), initiated migration but were delayed later in posterior migration and showed no dorsal migration. This phenotype is more severe than that of Egfr or Shc alone, suggesting that both RTKs might be affected. Prevention of PVR activity in double mutant cells did not block posterior migration, confirming that the requirement for these pathways was stage-specific and not EGFR-specific. Finally, analysis of mosaic clusters showed that Raf/MAPK and PLC-γ were important in late migration, reciprocal to the requirement for elmo. These results genetically define two migratory phases: an early posterior phase requiring ELMO-Mbc and a later posterior and dorsally directed phase requiring Raf/MAPK or PLC-γ. Both RTKs shift effector-pathway-dependency as migration progresses (Bianco, 2007).
To investigate the different migratory phases, border-cell migration was examined via live imaging. Appropriate conditions were establised for culturing and imaging of egg chambers, considering only active, growing ones. Border cells were selectively labelled with green fluorescent protein (GFP) and all membranes were labelled with the vital dye FM4-64. For all 24 wild-type samples, the identity of the front cell changed during the observation period, confirming the inference from fixed samples that cells change position during migration. This indicates that there is no determined front-cell fate. A clear difference was observed in behaviour of clusters during early (first half) and late phases. Early clusters had one, sometimes two, highly polarized cells clearly leading the migration; once these cells delaminated they moved straight and relatively fast. Weakly stained extensions protrude far from delaminating cells and subsequently shorten during movement, suggesting a 'grapple and pull' mechanism. Midway towards the oocyte, strong polarization was lost and cells rounded and started to 'shuffle' while dynamically probing the environment with short extensions. Occasionally the cluster would rotate or 'tumble' completely. This shuffling behaviour still provided effective movement of the cluster towards the oocyte and dorsally, albeit more slowly. Labelling cells with nuclear GFP allowed visualization of changes in positions within the cluster. The front cell exchanged, on average, every 18 min (Bianco, 2007).
As expected, positions corresponding to the second, slower phase of migration were more represented when cluster position along the migratory path was quantified in fixed samples. Also, border cells expressing dominant negative PVR and EGFR were individually active but provided little net cluster movement, as expected from the lack of guidance information. Finally, uniform overexpression of the attractant PVF1 caused an increased shuffling behaviour in the early phase but allowed slow forward movement, resembling normal late migration. This indicates that migrating clusters can interpret a shallow gradient when using the shuffling mode. It also suggests that the normal change in migratory behaviour midway into posterior migration might be triggered by the higher concentration of ligands closer to the oocyte (Bianco, 2007).
The early phase of migration with a highly polarized front cell corresponds temporally to the genetic requirement for ELMO activity. During the later phase, individual elmo mutant cells can alternate with wild-type cells in the lead position. Genetic analysis showed that Raf and MAPKK and, by inference, MAPK activation was sufficient to convey late guidance information. This was puzzling because MAPK activation appeared uniform in migrating border cells, and localized effects are usually a hallmark of guidance signalling. However, signalling that is not localized within an individual cell could still transmit spatial guidance information to the cell cluster if the cell with higher overall signalling indicates the direction of subsequent migration for the whole cluster, as observed for MAPK signalling in border cells. In this 'collective guidance' scenario, each cell of the cluster can be thought of as being analogous to a sector of an individual guided cell. Different levels of signalling in individual cells of the cluster transform into migration vectors because border cells adhere to each other and these contacts differ from substrate contacts. The occasional tumbling of border-cell clusters emphasizes the ability of these cells to behave as a collective unit. Tumbling may help single guided cells to 'reassess' their environment (Bianco, 2007).
To test this model for guidance, the relative levels of signalling in individual cells of the cluster were manipulated. Dynamic shuffling should allow cells to constantly 'compete' for the front position. None of the manipulations discussed below improved migration if all cells in a cluster were affected. Individual border cells with moderately elevated levels of PVR or EGFR were preferentially in the front relative to wild-type cells. Cells with elevated PVR tended to stay in or near the front position, suggesting that they were not competed away by other cells. This bias was ligand-dependent, because reducing PVF1 levels shifted the bias from PVR to EGFR, as was also shown by analysis of dorsal migration. Thus, increased signalling gives a cell-front bias when measuring an informative ligand. Elevating intracellular signalling levels had similar effects, whether by overexpression of an active form of Raf or by preventing downregulation of signalling as in Hrs mutant cells, in which RTK-mediated MAPK signalling is elevated in enlarged endosomes. The more modest front bias in Hrs mutant cells was reflected in behaviour: they could be displaced from the front. The E3 ubiquitin ligase Cbl negatively regulates RTK signalling and is also required to maintain localized RTK signalling within border cells initiating migration. Cbl mutant cells shifted from being preferentially at the back during early stages to being in the front during later migration. This indicates a transition from a mode requiring Cbl-dependent localization of signalling within the leading cell to a mode based on collective decisions within the cluster, in which Cbl mutant cells have an advantage owing to elevated RTK signalling (Bianco, 2007).
It is suggested that guidance of border-cell migration is achieved by two means: signalling localized within the cell, as used in individual migrating cells, and collective guidance, whereby the cluster uses differences in signalling strength among its constituent cells to determine direction. The two modes use the same guidance cues and receptors, but different downstream effectors. Localized signalling is required for the initial, polarized rapid migration, whereas collective behaviour, though observable throughout, dominates in the later phase. Collective decisions on the basis of differences in RTK signalling strength are important in Caenorhabditis elegans vulval development and in branching of Drosophila tracheal tubes, in which they result in specification of discrete cell fates. This differs from the dynamic situation reported in this study, in which the identity of the leading cell constantly changes. Indeed, the frequent exchange of leading cells suggests that front behaviour is normally temporarily restricted, possibly by induced inactivation of signalling. Such dynamics may allow the cluster to better reassess the environment. For guided migration of cell groups, this analysis indicates that sensing and regulation happens both at the single cell level and at the next level-that of collective cell decisions (Bianco, 2007).
Unravelling the molecular mechanisms that govern cell migration is of great importance towards understanding both normal embryogenesis and physiological and pathological processes occurring in the adult. Migration of border cells (BCs) during Drosophila oogenesis provides a simple and attractive model in which to address this problem. This study shows that the molecular chaperone Hsp70 is required for BC migration. Thus, BCs lacking all Hsp70 genes present in the fly genome fail to reorganize their actin cytoskeleton, resulting in migration defects. Similar defects are found when the Hsp70 co-chaperone DnaJ-1, the Drosophila homolog of the human Hsp40, is overexpressed specifically in BCs. In addition, biochemical and genetic evidence is provided for an interaction between DnaJ-1 and PDGF/VEGF receptor (PVR), which is also required for actin-mediated BC migration. Furthermore, the results showing that PVR also interacts genetically with Hsp70 suggest that a mechanism by which the DnaJ-1/Hsp70 chaperone complex regulates BC migration is by modulating PVR function (Cobreros, 2008).
The migration of the border cells (BCs) in the Drosophila egg chamber represents an excellent model system to dissect genetically the mechanisms regulating cell migration in vivo. BCs originate as a group of 6-10 somatic cells located at the anterior terminal pole of the ovarian follicular epithelium. BCs comprise 6-8 outer border cells and two anterior polar cells. At stage 9, BCs undergo a partial epithelial-mesenchymal transition (EMT), change their shape, delaminate from the follicular epithelium (FE) and become migratory as a cluster. By extending F-actin-based protrusions in between the nurse cells (NCs), outer BCs migrate posteriorly until they contact the anterior membrane of the oocyte at stage 10, carrying the non-motile polar cells with them . Once it reaches the oocyte, the BC cluster migrates dorsally to reside eventually at the anterior dorsal corner of the oocyte. Concomitant to BC migration, the main FE moves towards the posterior pole of the egg chamber (Cobreros, 2008).
A variety of molecules that regulate BC migration have been identified. The Drosophila homolog of the C/EBP transcription factor slow border cells (Slbo) is a key regulator of BC migration. It is expressed in BCs before and during their migration. Slbo is essential for BC motility; mutations in Slbo result in a failure in BC migration. However, Slbo is not sufficient to induce BC migration since earlier expression of Slbo in BCs does not cause a precocious migration. Slbo controls the expression of many genes required for BC migration including the Drosophila homolog of E-Cadherin (DE-Cad), which is crucial in both BCs and NCs for BC migration (Cobreros, 2008).
In addition, genetic screens have identified four signalling pathways that control spatially and temporally BC migration. Signalling through the JAK-STAT pathway is necessary and sufficient to convert stationary follicle epithelial cells into migratory and invasive cells. Thus, ectopic expression of the pathway in non-motile cells makes them migratory and mutations in different components of the pathway result in a failure to initiate migration. In addition, the JAK-STAT pathway is required for the recruitment of outer BCs by the anterior polar cells as inhibition of the JAK/STAT pathway results in a smaller number of BCs recruited to the cluster. Mutations in the Ecdysone hormone co-receptor taiman (tai) result in defects in BC migration even though Slbo expression is not altered. Furthermore, the current model proposes that the ecdysone hormone receptor pathway controls BC migration by regulating the turnover of DE-Cad-containing adhesive complexes. Finally, two Receptor Tyrosine Kinase (RTK) pathways, the Platelet-Derived Growth Factor/Vascular Endothelial Growth Factor Receptor (PVR) and the Epithelial Growth Factor Receptor (EGFR) pathways, have also been shown to control BC migration. In this case, as the expression of dominant negative forms of both receptors in BCs results in a delayed and misrouted migration, it has been proposed that these pathways act redundantly to guide BC migration (Cobreros, 2008 and references therein).
Despite the various genetic screens that have been conducted to isolate and characterize genes involved in BC migration, none of these studies have reached saturation and, as such, it is anticipated that a proportion of genes still remain to be identified. This is supported by recent studies that compared the transcriptome of migratory follicle cells with that of all other ovarian cells. The results of these studies revealed that the number of genes whose expression is significantly higher in migratory cells reaches several hundreds. These genes encode for ECM components and regulators as well as proteins involved in transcription, the regulation of the cytoskeleton, cell adhesion or signalling pathways. The myriad of functions performed by the identified genes reflects both the complexity of cell migration and the fact that there is still much to discover (Cobreros, 2008).
In order to isolate new elements required for the developmental regulation of BC migration, a gain-of-function screen was performed using an inducible system that allowed the overexpression of genes specifically in the adult thus avoiding deleterious gene expression during embryonic and larval stages. One of the genes which overexpression resulted in BC migration defects was DnaJ-1, the Drosophila homolog of the human co-chaperone Hsp40. DnaJ1/Hsp40 proteins have been conserved throughout evolution and are important for protein folding, translocation and degradation. They perform these roles primarily by regulating the activity of chaperone proteins, such as the heat-shock protein Hsp70, through stimulation of their ATPase activity. This work has investigated whether Hsp70 proteins are required for BC migration; removal of all Hsp70 genes present in the fly causes a delay in BC migration. In addition, biochemical and genetic interactions are described between DnaJ-1 and the PDGF- and VEGF-related receptor PVR, as well as a genetic interaction between PVR and Hsp70, suggesting that one mechanism by which DnaJ-1/Hsp70 could regulate BC migration is by modulating PVR function (Cobreros, 2008).
Hsp70 proteins are conserved molecular chaperones, found in the cytosol and in other compartments of the cell, that play an essential role in the life cycle of many proteins under both normal and stressful conditions. The house-keeping functions of Hsp70s include degradation of unstable and misfolded proteins, prevention and dissolution of protein aggregates, transport of proteins between cellular compartments, folding and refolding of proteins, uncoating of Clathrin-coated vesicles and control of regulatory proteins. In a conventional model for Hsp70's mechanism of action, the 'client' protein with exposed hydrophobic residues is first recognized and bound by the co-chaperone Hsp40, which delivers it to ATP-Hsp70. The J-domain of Hsp40 then triggers ATP hydrolysis and the locking of Hsp70 on the 'client', thus promoting its folding. 'Client' proteins whose activity is controlled through transient association with Hsp70 include regulatory proteins such as nuclear receptors (steroid hormone receptor), kinases (Raf, eIF2ξ-Kinase and CyclinB1) and transcription factors (HSF, c-Myc and pRb). Through these interactions, Hsp70 chaperones regulate important physiological processes such as cell cycle, cell differentiation or programmed cell death, as well as pathological processes such as oncogenesis, neurodegenerative diseases, viral infections and aging (Cobreros, 2008).
The work reported in this study reveals a novel role for the molecular chaperone Hsp70 in development: the regulation of border cell migration during Drosophila oogenesis. Interfering with Hsp70 function causes a delay in the migration of BCs. One of the initial steps in BC migration is an epithelial to mesenchymal transition, during which BCs reorganize their actin cytoskeleton, delaminate from the epithelium and begin to migrate between the nurse cells towards the oocyte. This study shows that removal of Hsp70 function results in a failure of F-actin redistribution at the leading front of motile cells. A role for Hsp70 and other chaperones, such as Hsp90, in F-actin reorganization has already been proposed from experiments in cell culture. In fact, incubation of Schwann cells with antibodies against Hsp90 leads to a rearrangement of the actin filaments present in the lamellipodia and to a reduction in the migrating ability of these cells. In addition, Hsp90 has also been involved in the actin-mediated motility of endothelial cells in vitro. Furthermore, both Hsp90 and the Hsp70 biding protein BAG-1 have been found to co-localize with F-actin during cell migration. Hsp70 could also act at other levels on the regulation of the actin cytoskeleton. Indeed, chaperones have been shown to perform many different and cooperative roles in the regulation of the cytoskeleton function (Liang, 1997). For instance, Hsp70 and Hsp90 have been shown to have an actin-binding activity that stabilizes the actin filaments by cross-linking. In addition, Hsp70 has also been shown to contribute to actin dynamics by assisting the chaperonin TriC on the folding of newly synthesized actin chains. Finally, the possibility cannot be ruled out that the effects of Hsp70 on actin reorganization are an indirect consequence of the role of Hsp70 on microtubules polymerization and assembly (Liang, 1997). However, preliminary results showing no effects on α-tubulin localization in Hsp70 mutant BCs do not support this possibility. Having shown that Hsp70 regulates BC migration, the use of a new technique that allows live imaging of actin dynamics during this process will help elucidate the role of these chaperones on actin cytoskeleton during cell migration in vivo (Cobreros, 2008).
The experiments reported in this study show that overexpression of DnaJ-1 results in BC migration defects. A shared feature of the DnaJ/Hsp40 family of proteins is that they all contain the J domain, which is responsible for the regulation of Hsp70 ATPase activity. In addition to this domain, many of the DnaJ/Hsp40 family members contain other conserved regions such as a Gly/Phe-rich region and/or cystein repeats. Depending on the presence of these other regions DnaJ proteins can be classified in three groups: Type I proteins harbour all three domains; Type II members possess the Gly/Phe-rich sequence but lack the cysteine repeats; finally, Type III proteins do not contain either of these two conserved regions. Besides their role in assisting Hsp70, type I and type II proteins, but not type III, can bind non-native substrates. However, in some cellular processes, such as the suppression of protein aggregation, while type I members can function independently of Hsp70, type II proteins must function in conjunction with Hsp70 to suppress aggregation. DnaJ-1 belongs to the Type II group, which suggests that it normally acts as a co-chaperone for Hsp70 proteins. In this context, overexpression of DnaJ-1 might result in over activation of Hsp70 that in turn can affect BC migration. This is supported by data showing that overexpression of Hsp70 also results in BC migration defects. However, the possibility cannot be discarded that during BC migration DnaJ-1 could also act on its own. In addition, DnaJ/Hsp40 has also been shown to regulate other chaperones, such as the Hsp90 proteins. The isolation of mutants in the DnaJ-1 gene should facilitate further investigation into the interrelationship between DnaJ-1 and Hsp70 in their novel role in BC cell migration (Cobreros, 2008).
The results also reveal a molecular and genetic interaction between DnaJ-1 and the PDGF/VEGF receptor, which is also required for actin cytoskeleton reorganization and cell morphology during border cell migration. This finding opens the possibility for the Hsp40-Hsp70 proteins to exert their function on BC migration partly through the regulation of the activity of PVR, which could in turn trigger intracellular signals leading to motility. In fact, it is becoming increasingly accepted that chaperone interactions serve a variety of functions that go beyond folding insufficiency, as some target proteins do not appear to be generally misfolded. Furthermore, a role for the chaperones Hsp70 and Hsp90 in the maturation of receptors of the steroid and estrogen families has already been shown. The current model is that several molecules of the chaperone machinery, including Hsp90, Hsp70 and Hsp40, associate with steroid receptors to form heterocomplexes. These interactions are, on one hand, required for the transition of the receptor into a high affinity state and, on the other, they appear to account for the repression of receptor function that is relieved upon hormone binding. In addition, chaperones can also regulate responses downstream of receptors by controlling their recycling or degradation. Thus, overexpression of Hsp70 has been shown to inhibit responses downstream of plasma membrane receptors, such as the insulin receptor, by preventing insulin receptor recycling, a mechanism that has been proposed to operate during heat stress to protect the receptor from thermal damage. In this scenario, one way it can be envisioned why overexpression or loss of function of components of the chaperone complex result in similar phenotypes during BC migration is the following. On one hand, loss of any of the components of the chaperone complex, such as Hsp70, would prevent full activation of PVR, leading to a failure in BC migration. On the other hand, overexpression of components of the chaperone complex, such as Hsp40 or Hsp70, could either sequester the receptor in the heterocomplex interfering with its release by ligand binding, or inhibit its recycling or promote its degradation. Alternatively, the effects of Hsp40 or Hsp70 overexpression in BC migration could also be independent of their interactions with the PVR pathway, and instead be related, for instance, to the role of Hsp proteins in actin stabilization mentioned above. Although at present these possibilities cannot be distinguish, the results show that Hsp70 and Hsp40 levels must be tightly regulated to allow proper BC migration (Cobreros, 2008).
Although there is increasing evidence in support of the role of molecular chaperones on cytoskeleton dynamics during stress, little is known about the involvement of this function in unstressed cells. This study has shown that the Hsp70 and DnaJ-1 are indeed required for the reorganization of the actin cytoskeleton in cell migration events occurring during development. Thus, the potent molecular and genetic tools available in Drosophila can now be used to decipher at the cellular and molecular level the mechanisms by which chaperones regulate cell migration in vivo. Furthermore, there is accumulating new evidence supporting a role for cell-surface chaperones on cancer metastasis. For instance, the expression of Hsp70 in human breast cancer cells has been correlated with metastasis and poor prognosis. Similarly, over expression of the Hsp70 binding protein BAG-1 accelerates cell motility of human gastric cancer cells. Thus, furthering understanding of the role of chaperones on cell migration will also help gain better understanding of the molecular mechanisms leading to metastatic spread, and to the identification of therapeutic methods to treat metastasis, one of primary cause of mortality associated with cancer (Cobreros, 2008).
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