InteractiveFly: GeneBrief

von Hippel-Lindau: Biological Overview | References

Mutations in the human von Hippel-Lindau (VHL) genes are the cause of VHL disease, which displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF). However, many other HIF-independent functions of VHL have been identified and recent evidence indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes. Many of these functions have not been verified in genetically tractable systems. Using an established follicular epithelial model in Drosophila, this study shows that the Drosophila VHL gene is involved in epithelial morphogenesis via stabilizing microtubule bundles and aPKC. Microtubule defects in VHL mutants lead to mislocalization of aPKC and subsequent loss of epithelial integrity. Destabilizing microtubules in ex vivo culture of wild-type egg chambers can also result in aPKC mislocalization and epithelial defects. Importantly, paclitaxel-induced stabilization of microtubules can rescue the aPKC localization phenotype in Drosophila VHL mutant follicle cells. The results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity (Duchi, 2010).

Establishing and maintaining epithelial integrity is essential for embryonic development, organogenesis and tissue remodeling. The key characteristic of epithelial cells is asymmetrical specification of membrane domains marked by domain-specific proteins. The epithelial morphogenic mechanism, although with some variations in different epithelial tissues, is highly conserved from worm to mammal. The crucial initial step in establishing epithelial polarity is the specification of the apical domain, which is defined by the function of a complex containing atypical PKC (aPKC), Bazooka (Baz; mammalian and worm PAR-3) and PAR-6. The PAR complex is initially recruited by activated Cdc42 to the apical domain. The three proteins were originally thought to function as a complex; however, recent evidence indicates that Baz might be required first to recruit the aPKC-PAR-6 complex to the subapical domain juxtaposed to the future adherens junction (AJ) (Harris, 2007). The PAR complex is required for the localization of another apical complex containing Crumbs (Crb), Stardust (Std; mammalian Pals1) and Discs lost (Dlt; mammalian Patj). The PAR- and Crb-containing complexes occupy the apical-most region of the lateral membrane, just apical to the AJs (Duchi, 2010).

The apical complexes in turn restrict the localization of a third complex comprising Scribble (Scrib; mammalian Scribble/Vartul), Discs large (Dlg) and Lethal(2) giant larvae (Lgl) to the basolateral domain, while Lgl also antagonizes the apical components and prevents their spreading to the basolateral side. The antagonistic action of apical and basolateral complexes helps define the apicolateral loci eventually occupied by AJs. It is not yet completely clear how the initial localization of the apical complex is achieved (Duchi, 2010).

The VHL tumor-suppressor gene mutations are the genetic cause of the familial VHL disease. Germline mutations in VHL predispose the patients to several benign and malignant tumors, including renal cell carcinoma (RCC, kidney cancer), hemangioblastoma (overgrowth of blood vessels in the retina and central nervous system) and pheochromocytoma (tumors in the adrenal glands). VHL protein has been shown to function as an E3 ubiquitin ligase. Among its best-documented targets is the alpha subunit of the hypoxia-inducible factor (HIF-α). Therefore, the canonical tumor-suppressor function of VHL is modulation of the normal oxygen-sensing mechanism that regulates angiogenic response and metabolic switch to glycolysis (Kaelin, 2008a). However, how this function correlates with the origin of epithelial tumors such as RCC is unclear (Frew, 2007), although it is thought that HIF-independent mechanisms might be involved (Duchi, 2010).

VHL is evolutionarily conserved. In Drosophila, the VHL gene has been implicated in tracheal tubule development and HIF-α regulation in the embryos based on biochemical and RNA interference-mediated phenotypic studies (Adryan, 2000; Arquier, 2006; Aso, 2000; Mortimer, 2009). In this report, the first genomic Drosophila VHL mutant was generated, and the function of VHL in epithelial morphogenesis was examined using a model epithelium -- the follicle cells in the egg chamber. VHL is shown to regulate the proper localization and stability of aPKC in the follicle cells, and this function is, at least in part, mediated by the action of VHL on microtubule (MT) stability. Without VHL function, MTs and aPKC are destabilized, resulting in epithelial defects. These results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity (Duchi, 2010).

The VHL mutation was generated using the homologous recombination strategy. Homozygous VHL¹ mutants are sluggish after hatching and die at the end of first instar larval stage. Wild-type, heterozygous and homozygous first instar larvae were hand-picked and subjected to genomic DNA PCR. The homozygous mutant animals show complete loss of the wild-type gene copy. The VHL¹ allele, when paired with a deficiency chromosome encompassing the VHL locus (at 47E), shows the same late first instar lethality, suggesting that VHL¹ is a null mutant. The lethal phenotype can be rescued by expressing a wild-type VHL cDNA under the control of the hsp70 promoter. Therefore, VHL gene truncation is the only major genetic defect in the VHL¹ allele (Duchi, 2010).

The Drosophila follicle cells exhibit the typical epithelial polarity exemplified by markers such as the apical PAR complex, AJ components and the basolateral Lgl complex. Establishment of the epithelial polarity begins soon after follicle cells diverge from the somatic stem cells and encircle the germ cells (stage 1). The epithelium reaches maturity at mid-oogenic stages (after stage 6 at ~30 hours of egg chamber development; total developmental time ~70 hours), when follicle cells cease to proliferate. To examine the phenotype in the follicular epithelium, an adult tissue, mosaic mitotic mutant clones were generated using the Flp/FRT system. Mutant clones are identified by a lack of GFP expression. In an initial survey of potential egg chamber phenotypes, prominent epithelial defects were observed at stage 10 (about 50-55 hours into egg chamber development. The most notable morphological abnormalities were the piling-up of follicle cells and stretched follicular epithelium. The same phenotypes were observed with the VHL deficiency Df(2R)en-A. To quantify the phenotypes, 100 clones of various sizes at stage 9-10 were analyzed. Clone sizes were categorized based on the number of cells in an optical cross-section. As the penetrance in single-cell clones is variable, probably because of phenotypic rescue by the neighboring epithelial cells, only clones larger than two cells were considered. Thirty-eight percent of clones showed stacking of follicle cells and the more severe, multilayering phenotypes (more than three layers of cells); 42% showed flattening/stretching of epithelium; and the remaining 20% showed swelling. It was also determined that VHL loss-of-function mutation did not affect cell proliferation or viability. As mitotic recombination in a heterozygous progenitor cell (VHL¹/ubi-GFP) generates one VHL homozygote (no GFP) and one wild type (two copies of GFP), the numbers of high GFP-expressing cells and GFP-negative cells should be equal if the mutant cells do not exhibit an altered rate of proliferation or viability. The ratio was calculated of the cell number within the VHL¹ mutant clones versus the cell number within the sister wild-type clones, and a mean value of 0.96 was obtained. Importantly, the ratio between the cell number in mutant and wild-type sister clones did not change as a function of the clone size. Furthermore, staining for mitosis and apoptosis markers phospho-histone H3 and cleaved caspase 3, respectively, also showed that VHL mutation does not affect cell proliferation or caspase-mediated apoptosis (Duchi, 2010).

This report shows that Drosophila VHL is important for establishing and maintaining epithelial integrity via its regulation of MT and aPKC stability. MT disruption and epithelial phenotypes were observed early in oogenesis. This indicates that MT bundles in developing epithelial cells are crucial for epithelial development and are under pressure from dynamic instability. Without stabilizing activity provided by VHL, MTs are disorganized and ultimately disintegrate, resulting in loss of epithelial integrity. Disrupted MTs interfere with proper localization of aPKC, which in turn leads to mislocalization of downstream epithelial markers and epithelial defects. An ex vivo experiment also demonstrates that epithelial defects can occur within a short time (relative to the entire oogenesis time frame) after destabilizing MTs in non-proliferating epithelial cells. This indicates that the maintenance of epithelial integrity is a dynamic and continuous process even in a stable epithelium, for which MTs are crucially important. Previous studies using RNA interference-mediated knockdown demonstrated a morphogenic role of VHL in trachea development (Adryan, 2000; Mortimer, 2009). The tracheal phenotypes appear to be the result of elevated cell motility and ectopic chemotactic signaling. Therefore, the tracheal function of VHL might be mediated via different VHL targets in a tissue-specific context. Alternatively, regulation of MT stabilization might also be the underlying mechanism. A separate, tissue-specific function for VHL is favored since the tracheal defects in VHL knockdown can be relieved by decreased expression of breathless, which encodes the chemotactic signaling receptor in the trachea. The two VHL functions, however, are not necessarily mutually exclusive. These different organ systems might in the future serve as a model for testing whether the various functions assigned to VHL are tissue-specific and context-dependent (Duchi, 2010).

Human VHL has been shown to translocate aPKC to MTs, thereby influencing MT reorganization (Schermer, 2006). This study shows that the aPKC mutant can affect MT organization but not stability, whereas VHL can influence both. Conversely, disruption of MTs alone can result in aPKC mislocalization resembling that observed in VHL mutant cells. Importantly, paclitaxel-induced MT stabilization can rescue aPKC localization in VHL mutant follicle cells. It is therefore concluded that a major function of VHL in the follicular epithelium is regulation of MT stability. Loss of MTs leads to aPKC mislocalization and degradation. Conversely, the results also indicate that part of the VHL epithelial functions might be mediated by its direct effect on aPKC stability, as exogenously expressed aPKC-GFP fusion protein can partially rescue (not statistically significant) the VHL mutant phenotype. Indeed, it was also demonstrated that VHL can co-immunoprecipitate with tubulin or aPKC, and that, at least in S2 cells, aPKC levels can be affected by VHL levels without affecting tubulin. Taken together, it appears that the epithelial function of VHL is mediated through stabilization of MT, with an auxiliary role in directly stabilizing aPKC (Duchi, 2010).

It has been suggested that VHL interacts with MTs via the kinesin 2 family of motors (Lolkema, 2007). Future studies using the Drosophila epithelial system should also address this issue in vivo. Also interestingly, this study showed that the YH mutant can associate with MT but has little MT-stabilizing activity. This suggests that the YH mutant might be defective in recruiting other proteins, possibly including aPKC, that are important for regulating MT functions. In light of the role of Drosophila VHL in regulating MT stability, a function presumably important for all cells, it is curious that the tissue-specific btl-driven VHL expression can rescue the homozygous lethality of VHL¹. This study has shown that tracheal defects are the major embryonic phenotype observed in VHL mutant (Adryan, 2000). In the course of attempting to rescue the tracheal phenotype with btl-driven VHL, the appearance of rescued homozygous adults was noted. This indicates that the MT stabilizing function of VHL is not required in all tissues. It is possible that although VHL can enhance MT stability, by itself it is not an essential factor for MT polymerization. As such, some tissues might be less dependent on VHL levels. In the follicular environment, MT rearrangement, including depolymerization and repolymerization, is crucial when the entire epithelial sheet moves over the germ cell complex while the cells grow increasingly columnar. MT stabilization facilitated by VHL might be of particular importance during this process (Duchi, 2010).

The best-documented function of VHL is its E3 ubiquitin ligase activity that targets the alpha subunit of the HIF transcription factor. This activity provides an elegant mechanistic explanation for the hypervascularity of many of the VHL tumors and for a potential contributor to the metabolic switch to glycolysis, as HIF can upregulate pro-angiogenic factors such as vascular-endothelial growth factor and components in the glucose metabolic pathway. However, recent evidence has suggested that VHL is a multifunctional protein. It can function as a regulator of matrix deposition (Ohh, 1998), integrin assembly, endocytosis (Champion, 2008; Hsu, 2006), kinase activity (Yang, 2007), senescence (Young, 2008), protein stabilities (Chitalia, 2008; Roe, 2006) and tight junction formation (Calzada, 2006; Harten, 2009), among many others (Frew, 2007). Whether tight junction disassembly in VHL mutant cells is HIF-dependent is still unresolved; however, other HIF-independent functions appear to facilitate protein stability or activity instead of destabilizing them as a ubiquitin ligase. Such chaperon/adaptor function has also been implicated in promoting stability of MTs (Hergovich, 2003; Lolkema, 2004). The MT-stabilizing function, although potentially highly significant, has so far only been linked to cilium biogenesis and mitotic spindle orientation in cultured RCC and renal tubule cells (Lolkema, 2008; Lutz, 2006; Schermer, 2006; Thoma, 2009). The physiological and developmental significance of this function has not been elucidated in vivo. Indeed, it is unclear how loss of many of these HIF-independent functions contributes to VHL tumor formation because of a lack of tractable genetic models (Duchi, 2010).

One crucial element in tumorigenesis is the breakdown of epithelial integrity that ultimately leads to epithelial-to-mesenchymal transition. This report provides the first demonstration of a potential tumor-suppressor function for VHL in regulating epithelial morphogenesis via its role in promoting MT stability. Future studies should exploit further this genetic system for elucidating how a myriad of disease-related VHL point mutations might differentially influence such function (Duchi, 2010).

Dose-dependent modulation of HIF-1alpha/sima controls the rate of cell migration and invasion in Drosophila ovary border cells

The role of the hypoxic response during metastasis was analysed in migrating border cells of the Drosophila ovary. Acute exposure to 1% O₂ delayed or blocked border cell migration (BCM), whereas prolonged exposure resulted in the first documented accelerated BCM phenotype. Similarly, manipulating the expression levels of sima, the Drosophila hypoxia-inducible factor (HIF)-1alpha ortholog, revealed that Sima can either block or restore BCM in a dose-dependent manner. In contrast, over-expression of Vhl (Drosophila von Hippel-Lindau) generated a range of phenotypes, including blocked, delayed and accelerated BCM, whereas over-expression of hph (Drosophila HIF prolyl hydroxylase) only accelerated BCM. Mosaic clone analysis of sima or tango (HIF-1beta ortholog) mutants revealed that cells lacking Hif-1 transcriptional activity were preferentially detected in the leading cell position of the cluster, resulting in either a delay or acceleration of BCM. Moreover, in sima mutant cell clones, there was reduced expression of nuclear slow border cells (Slbo) and basolateral DE-cadherin, proteins essential for proper BCM. These results show that Sima levels define the rate of BCM in part through regulation of Slbo and DE-cadherin, and suggest that dynamic regulation of Hif-1 activity is necessary to maintain invasive potential of migrating epithelial cells (Doronkin, 2010).

Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia

The tracheal system of Drosophila is an interconnected network of gas-filled epithelial tubes that develops during embryogenesis and functions as the main gas-exchange organ in the larva. Larval tracheal cells respond to hypoxia by activating a program of branching and growth driven by HIF-1α/sima-dependent expression of the breathless (btl) FGF receptor. By contrast, the ability of the developing embryonic tracheal system to respond to hypoxia and integrate hard-wired branching programs with sima-driven tracheal remodeling is not well understood. This study shows that embryonic tracheal cells utilize the conserved ubiquitin ligase dVHL to control the HIF-1 α/sima hypoxia response pathway, and two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes were identified: a relatively hypoxia-resistant 'early' phase during which Sima activity conflicts with normal branching and stunts migration, and a relatively hypoxia-sensitive 'late' phase during which the tracheal system uses the dVHL/sima/btl pathway to drive increased branching and growth. Mutations in the archipelago (ago) gene, which antagonizes btl transcription, re-sensitize early embryos to hypoxia, indicating that their relative resistance can be reversed by elevating activity of the btl promoter. These findings reveal a second type of tracheal hypoxic response in which Sima activation conflicts with developmental tracheogenesis, and identify the dVHL and ago ubiquitin ligases as key determinants of hypoxia sensitivity in tracheal cells. The identification of an early stage of tracheal development that is vulnerable to hypoxia is an important addition to models of the invertebrate hypoxic response (Mortimer, 2008).

The development and survival of an organism are dependent on its ability to adapt to changing environmental conditions. Responses to some environmental changes, for example in nutrient availability, temperature, or oxygen concentration, involve alterations in patterns of gene expression that allow the organism to survive periods of environmental stress. In metazoan cells, the cellular response to reduced oxygen is mediated primarily by the HIF (hypoxia inducible factor) family of transcription factors, which are heterodimers composed of α and β subunits belonging to the bHLH Per-ARNT-Sim (bHLH-PAS) protein family. The HIF-1 αβ heterodimer is the primary oxygen-responsive HIF in mammalian cells and binds to a specific DNA sequence termed hypoxia response element (HRE) present in the promoters of target genes involved in energy metabolism, angiogenesis, erythropoiesis, and autophagy. HIF-1 activity is inhibited under normoxic conditions by two hydroxylase enzymes that use dioxygen as a substrate for catalysis to hydroxylate specific proline or aspartate residues in the HIF-1α subunit (reviewed in Kaelin, 2008b). These modifications limit HIF-1 activity by either reducing HIF-1α levels or inhibiting its ability to activate HRE-containing target promoters. One of these inhibitory mechanisms involves the 2-oxoglutarate/Fe(II)-dependent HIF-1 prolyl hydroxylase (HPH), which attaches a hydroxyl group onto each of two conserved proline residues in the oxygen-dependent degradation domain (ODD) of mammalian HIF-1α. These modifications create a binding site in the HIF-1α ODD for the Von Hippel-Lindau (VHL) protein, the substrate adaptor component of a ubiquitin ligase that subsequently polyubiquitinates HIF-1α and targets it for degradation by the proteasome (reviewed in Kaelin, 2005). This degradation mechanism operates constitutively in normoxia and is epistatic to otherwise wide spread expression of HIF-1α mRNA. HIF-1α protein is also modified by a second oxygen-dependent hydroxylase termed Factor Inhibiting HIF (FIH) that hydroxylates an asparagine residue in the HIF-1α C-terminal activation domain (reviewed in Kaelin, 2005). This blocks interaction with the CBP/p300 transcriptional co-factor and thus further restricts expression of HIF-1 responsive genes. These parallel O₂-dependent hydroxylation mechanisms by HPH and FIH ensure that HIF-1α levels and activity remain low in normoxic conditions. However as oxygen levels become limiting in the cellular environment, rates of hydroxylation decline and HIF-1α is rapidly stabilized in a form that dimerizes with HIF-1β, translocates to the nucleus, and promotes transcription of HRE-containing target genes (Mortimer, 2008).

Evidence suggests that invertebrate homologs of HIF-1 are also regulated in response to changes in oxygen availability (reviewed by Gorr, 2006). In the fruit fly Drosophila melanogaster, the HPH homolog fatiga (fga) has been shown to genetically antagonize the HIF-1α homolog similar (sima) during development (Centanin, 2005). The Drosophila VHL homolog dVHL has also been shown to be capable of binding to human HIF-1α and stimulating its proteasomal turnover in vitro (Aso, 2000). In addition, the Drosophila genome encodes a well-characterized HIF-1β homolog tango (tgo), and two potential FIH homologs (CG13902 and CG10133; Berkeley Drosophila Genome Project) that have yet to be analyzed functionally. Spatiotemporal analysis of sima activation using sima-dependent hypoxia-reporter transgenes has shown that exposure to an acute hypoxic stress induces Sima most strongly in cells of the larval and embryonic tracheal system, while induction of reporter activity in other tissues requires more chronic exposure to low oxygen. The larval tracheal system is composed of an interconnected network of polarized, epithelial tubes that duct gases through the organism. As the trachea acts as the primary gas-exchange organ in the larva, it is thus a logical site of hypoxia sensitivity. During larval stages, specific cells within the tracheal system called 'terminal cells' respond to hypoxia by initiating new branching and growth that results in the extension of fine, unicellular, gas-filled tubes toward hypoxic tissues in a manner somewhat analogous to mammalian angiogenesis . Studies have shown that sima and its upstream antagonist fga function within terminal cells to regulate this process (Centanin, 2008). sima is necessary for terminal cell branching in hypoxia and its ectopic activation, by either transgenic overexpression or loss of fga, is sufficient to induce excess branching even in normoxia. These phenotypes have been linked to the ability of sima to promote expression of the breathless (btl) gene (Centanin, 2008), which encodes an FGF receptor that is activated by the branchless (bnl) FGF ligand. This receptor/ligand pair is known to act via a downstream MAP-kinase signaling cascade to promote cell motility and tubular morphogenesis in a variety of systems. Excessive activation of this pathway within tracheal cells by transgenic expression of btl is sufficient to drive excess branching. Reciprocally, misexpression of the bnl ligand in certain peripheral tissues is sufficient to attract excess terminal cell branching. Indeed production of secreted factors such as Bnl may be a significant part of the physiologic mechanism by which hypoxic cells attract new tracheal growth. Sima-driven induction of btl in conditions of hypoxia thus allows larval terminal cells to enter what has been termed an 'active searching' mode (Centanin, 2008) in which they are hyper-sensitized to signals emanating from nearby hypoxic non-tracheal cells (Mortimer, 2008 and references therein).

The role of the btl/bnl pathway in tracheal development is not restricted to hypoxia-induced branching of larval terminal cells. It also plays a critical, earlier role in the initial development of the embryonic tracheal system from the tracheal placodes, groups of post-mitotic ectodermal cells distributed along either side of the embryo that undergo a process of invagination, polarization, directed migration, and fusion to create a network of primary and secondary tracheal branches . btl and bnl are each required for this process via a mechanism in which restricted expression of bnl in cells outside the tracheal placode represents a directional cue for the migration of btl-expressing cells within the placode. Accordingly, btl expression is normally highest in pre-migratory and migratory embryonic fusion cells. In contrast to the larval hypoxic response, sima does not appear to be required for morphogenesis of the embryonic tracheal system. Rather, developmentally programmed signals in the embryo dictate a stereotyped pattern of btl and bnl expression that leads to a similarly stereotyped pattern of primary and secondary tracheal branches. The btl/bnl pathway thus responds to developmental signals to drive a fixed pattern of branching in the embryo, while in the subsequent larval stage it responds to hypoxia-dependent sima activity to facilitate the homeostatic growth of larval terminal cells and tracheal remodeling (Mortimer, 2008 and references therein).

Under normal circumstances, developing Drosophila tissues do not begin to experience hypoxia until the first larval stage, when organismal growth and movement begin to consume more oxygen than can be provided by passive diffusion alone. As a consequence, the first hypoxic challenge normally occurs after the btl/bnl-dependent elaboration of the primary and secondary embryonic branches is complete. Thus, the ability of the larval tracheal system to drive new branching and remodeling via sima and btl represents the response of a developed 'mature' tracheal system to reduced oxygen availability. By contrast the effect of hypoxia on embryonic tracheal development, which requires tight spatiotemporal control of Btl signaling to pattern the tracheal network, is not as well understood. Given that the trachea does not function as a gas-exchange organ until after fluid is cleared from the tubes at embryonic stage 17, it may be that the transcriptional response of embryonic tracheal cells to hypoxia leads to mainly metabolic changes rather than to a btl-driven program of tubulogenesis and remodeling. However, if the embryonic tracheal system does utilize the sima pathway to induce hypoxia-dependent changes in btl gene transcription, then hypoxic exposure of embryos might be predicted to produce a situation of competing developmental and homeostatic inputs that converge on the btl/bnl pathway. The ability of tracheal cells to integrate such signals may then determine whether or not the embryonic tracheal system is able to adapt to oxygen stress, or whether embryonic tracheal development represents a sensitive period during which the organism's ability to respond to changes in oxygen levels is inherently limited by a pre-programmed pattern of developmental gene expression (Mortimer, 2008).

This study shows that the embryonic tracheal system utilizes the dVHL/sima pathway to respond to hypoxia, but that the type and severity of resulting phenotypes depend on the developmental stage of exposure. Hypoxic challenge while embryonic tracheal cells are responding to developmentally programmed btl/bnl migration signals disrupts tracheal development and results in fragmented and unfused tracheal metameres. In contrast, hypoxic challenge at a somewhat later embryonic stage after fusion is complete results in overgrowth of the primary tracheal branches and the production of extra secondary branches. Interestingly, it was found that the threshold of hypoxia required to induce tracheal phenotypes in the early embryo is higher than that required to induce excess branching phenotypes in later embryonic stages, indicating that tracheal patterning events in the embryo are relatively resistant to hypoxia. Genetic analysis indicates that both types of hypoxic tracheal phenotypes -- stunting and overgrowth -- require sima and can be phenocopied in normoxia by reducing expression of the HIF-1α ubiquitin ligase gene dVHL specifically within tracheal cells. Moreover, it was found that reduced dVHL expression in the larval trachea leads to excess terminal cell branching in a manner quite similar to that observed in fga mutants. Molecular and genetic data indicate that excess btl transcription is a major cause of hypoxia-induced tracheal phenotypes. Consistent with this, mutations in the archipelago (ago) gene, which antagonizes btl transcription in tracheal fusion cells, synergize strongly with dVHL inactivation to disrupt tracheal migration and branching. Interestingly, ago mutations also lower the threshold of hypoxia required to elicit tracheal phenotypes in the 'early' embryo, suggesting that the relative activity of the btl promoter can affect hypoxic sensitivity. These findings show that the dVHL/sima pathway plays an important role in tracheal development, and identify two distinct phases of embryonic development that show different phenotypic outcomes of activating this pathway: an early phase during which sima activity conflicts with developmental control of tracheal branching and migration, and a later phase during which the tracheal system uses the dVHL/sima/btl pathway to adapt to hypoxia by increasing its future capacity to deliver oxygen to target tissues (Mortimer, 2008).

Hypoxia-induced remodeling of tracheal terminal cells represents the response of a developed larval tracheal system to reduced levels of O₂ in the environment. By contrast, the response of the developing embryonic tracheal system to systemic hypoxia has not been as well characterized. In light of the observation that embryonic tracheal cells display hypoxia-induced activation of a Sima-reporter) and that sima promotes btl expression in larval tracheal cells, embryonic exposure to hypoxia may thus produce a situation in which hard-wired btl/bnl patterning signals in the embryo come into conflict with the type of sima/btl-driven plasticity of tracheal cell branching seen in the larva. This study examined the effect of hypoxia on embryonic tracheal branching and migration. It was found that hypoxia has dramatic effects on the patterns of morphogenesis of the primary and secondary tracheal branches. Surprisingly, varying the timing and severity of hypoxic challenge is able to shift the outcome from severely stunted tracheal branching to excess branch number and enhanced branch growth. Genetic and molecular data indicate that both classes of phenotypes, stunting and overgrowth, involve regulation of sima activity and btl transcription by dVHL, and that the effects of hypoxia on tracheal development can be mimicked in normoxia by tracheal-specific knockdown of dVHL. This observation confirms a central role for dVHL in restricting the hypoxic response in vivo, and identifies a role for dVHL as a required inhibitor of sima and btl during normal tracheogenesis (Mortimer, 2008).

Since Trh and Sima/HIF-1α share a similar consensus DNA binding site, it is likely that the tracheal phenotypes elicited by either hypoxia or dVHL knockdown are to some degree the product of a combined 'Trh/Sima-like' transcriptional activity in tracheal cells. This conclusion is supported both by the general phenotypic similarity (i.e. migration and overgrowth defects) between hypoxia/dVHL knockdown and trh overexpression (Mortimer, 2007), by the modest ability of trh alleles to suppress dVHLⁱ phenotypes, and by the previously demonstrated overlap of transcriptional activity between Trh and human HIF-1α. Indeed, Trh is well-established as a required activator of developmental btl expression. However, because the excess Btl activity that occurs in hypoxia or in the absence of dVHL occurs independently of a change in Trh expression, it thus appears to be mediated largely by increased sima activity (Mortimer, 2008).

This analysis suggests that there are two distinct developmental 'windows' of embryogenesis during which hypoxia has opposite effects on tracheal branching. The first corresponds to a period immediately before and during primary branch migration that is relatively insensitive to hypoxia. Embryos in this stage show a minimal response to 1% O₂, but show a nearly complete arrest of migration in 0.5% O₂. Interestingly, a prior study found that similarly staged embryos (stage 11) respond to complete anoxia by prolonged developmental arrest, from which they can emerge and resume normal development. These somewhat paradoxical results -- that acute hypoxia is more detrimental to development than chronic anoxia -- might be explained by the observation that chronic exposure to low O₂ induces Sima activity throughout the embryo while acute exposure activates Sima only in tracheal cells. The former scenario may result in coordinated developmental and metabolic arrest throughout the organism, while in the latter scenario developmental patterns of gene expression in non-tracheal cells may proceed such that tracheal cells emerging from an 'early' hypoxic response find an embryonic environment in which developmentally hard-wired migratory signals emanating from non-tracheal cells have ceased (Mortimer, 2008).

The second type of tracheal response occurs during a later 'window' of embryogenesis after btl/bnl-driven primary and secondary branch migration and fusion are largely complete. It involves sinuous overgrowth of the primary and secondary branches, and duplication of secondary branches. As in the 'early' response, 'late' hypoxic phenotypes are controlled by the dVHL/sima pathway, yet unlike the 'early' response, these phenotypes occur at high penetrance even at 1% O₂. Thus the 'late' embryonic tracheal system is relatively sensitized to hypoxia and responds with increased branching in a manner similar to larval terminal cells. Indeed, much as larval branching increases with decreasing O₂ levels, it was observed that dorsal trunk growth in the late embryo is graded to the degree of hypoxia. The mechanism underlying the differential sensitivity of the 'early' and 'late' tracheal system may be quite complex. However, it was found that tracheogenesis can be sensitized to hypoxia by reducing activity of ago, a ubiquitin ligase component that restricts btl transcription in tracheal cells via its role in degrading the Trh transcription factor (Mortimer, 2007). Increasing transcriptional input on the btl promoter thus appears to sensitize 'early' tracheal cells to hypoxia. As Sima also controls btl transcription, one explanation of the difference in sensitivity between different embryonic stages may thus lie in differences in the activation state of the btl promoter. If so then the activity of the endogenous btl regulatory network may be an important determinant of the threshold of hypoxia required to elicit changes in tracheal architecture (Mortimer, 2008).

An organism can have its hypoxic response triggered in two ways, either by systemic exposure of the whole organism to a reduced O₂ environment or by localized hypoxia produced by increased O₂ consumption in metabolically active tissues. Data from this study and others (Centanin, 2008; Jarecki, 1999) suggests there may be distinctions between these two triggers. Exposing larvae or embryos to a systemic pulse of hypoxia results in a 'btl-centric' response specifically in tracheal cells. Outside of an 'early' vulnerable period which corresponds to embryonic branch migration and fusion, elevated Btl activity in embryonic tracheal cells promotes branch duplications and overgrowth similar to that seen in larvae (Centanin, 2008). By contrast, tracheal growth induced by localized hypoxia in the larva has been suggested to involve a 'bnl-centric' model in which the hypoxic tissue secretes Bnl and recruits new tracheal branching (Jarecki, 1999). Whether this type of mechanism operates in embryos, or whether embryos ever experience localized hypoxia in non-tracheal cells, has not been established (Mortimer, 2008).

tHE data indicate that dVHL is a central player in the hypoxic response pathway in embryonic and larval tracheal cells. A prior study found that injection of dVHL dsRNA into syncytial embryos disrupted normal tracheogenesis (Adryan, 2000), but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. The current study found that dVHL knockdown specifically in tracheal cells mimics the effect of systemic hypoxia on embryonic tracheal architecture and larval terminal cell branching. dVHL knockdown thus phenocopies loss of the HPH gene fga, which normally functions to target Sima to the dVHL ubiquitin ligase in normoxia (Centanin, 2005). Moreover, all phenotypes that result from reduced dVHL expression can be rescued by reducing sima activity, suggesting that Sima is the major target of dVHL in the tracheal system. These data support a model in which dVHL, fga, and sima function as part of a conserved VHL/HPH/HIF-1α pathway to control tracheal morphogenesis in embryos and larvae. The btl receptor appears to be an important target of this pathway in embryonic (this study) and larval (Centanin, 2008) tracheal cells. Knockdown of dVHL elevates btl transcription in embryonic placodes and tracheal branches, and removal of a copy of the gene effectively suppresses dVHL tracheal phenotypes. Reciprocally, overexpression of wild type btl in embryonic tracheal cells can produce migration defects and sinuous overgrowth (this study; Mortimer, 2007), while expression of a constitutively active btl chimera (btl^λ) also leads to primary branch stunting and duplication of secondary branches. Interestingly, pupal lethality associated with tracheal-specific knockdown of dVHL is not sensitive to the dose of btl, but is dependent on sima. Thus the dVHL/sima pathway may have btl independent effects on tracheal cells in later stages of development (Mortimer, 2008).

von Hippel-Lindau tumor suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish

Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the development of highly vascularized neoplasms in multiple organ systems. This study shows that zebrafish vhl mutants display a marked increase in blood vessel formation throughout the embryo, starting at 2 days post-fertilization. The most severe neovascularization is observed in distinct areas that overlap with high vegfa mRNA expression, including the vhl mutant brain and eye. Real-time quantitative PCR revealed increased expression of the duplicated VEGFA orthologs vegfaa and vegfab, and of vegfb and its receptors flt1, kdr and kdr-like, indicating increased vascular endothelial growth factor (Vegf) signaling in vhl mutants. Similar to VHL-associated retinal neoplasms, diabetic retinopathy and age-related macular degeneration, this study showed, by tetramethyl rhodamine-dextran angiography, that vascular abnormalities in the vhl^-/- retina leads to vascular leakage, severe macular edema and retinal detachment. Significantly, vessels in the brain and eye express cxcr4a, a marker gene expressed by tumor and vascular cells in VHL-associated hemangioblastomas and renal cell carcinomas. VEGF receptor (VEGFR) tyrosine kinase inhibition (through exposure to sunitinib and 676475) blocked vhl^-/--induced angiogenesis in all affected tissues, demonstrating that Vegfaa, Vegfab and Vegfb are key effectors of the vhl^-/- angiogenic phenotype through Flt1, Kdr and Kdr-like signaling. Since it was shown that the vhl^-/- angiogenic phenotype shares distinct characteristics with VHL-associated vascular neoplasms, zebrafish vhl mutants provide a valuable in vivo vertebrate model to elucidate underlying mechanisms contributing to the development of these lesions. Furthermore, vhl mutant zebrafish embryos carrying blood vessel-specific transgenes represent a unique and clinically relevant model for tissue-specific, hypoxia-induced pathological angiogenesis and vascular retinopathies. Importantly, they will allow for a cost-effective, non-invasive and efficient way to screen for novel pharmacological agents and combinatorial treatments (van Rooijen, 2010).

von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina

In early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. This process is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. This study seeks other mechanisms that regulate this process, and focuses on the dramatic change in oxygen environment at the point of birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor alphas (HIF-αs) under normoxic, but not hypoxic, conditions. To examine the role of oxygen-sensing mechanisms in retinal circulatory system transition, retina-specific conditional-knockout mice for VHL (Vhl^α-CreKO mice) were generated. These mice exhibit arrested transition from the fetal to the adult circulatory system, persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelial growth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1αα gene. These results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the fetal to the adult circulatory system in the retina (Kurihara, 2010).

Analysis of the hypoxia-sensing pathway in Drosophila melanogaster

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. The present study provides direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), the hypoxic cascade was analyzed in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia (Arquier, 2006).

Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to a variety of human tumors, including renal cell carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. This study reports on the identification and characterization of the Drosophila homolog of VHL. The predicted amino acid sequence of Drosophila VHL protein shows 29% identity and 44% similarity to that of human VHL protein. Biochemical studies have shown that Drosophila VHL protein binds to Elongins B and C directly, and via this Elongin BC complex, associates with Cul-2 and Rbx1. Like human VHL, Drosophila VHL complex containing Cul-2, Rbx1, Elongins B and C, exhibits E3 ubiquitin ligase activity. In addition, evidence is provided that hypoxia-inducible factor (HIF)-1alpha is the ubiquitination target of both human and Drosophila VHL complexes (Aso, 2000).

Tracheal development and the von Hippel-Lindau tumor suppressor homolog in Drosophila.

von Hippel-Lindau disease is a hereditary cancer syndrome. Mutations in the VHL tumor suppressor gene predispose individuals to highly vascularized tumors. However, VHL-deficient mice die in utero due to a lack of vascularization in the placenta. To resolve the contradiction, the Drosophila VHL homologue (d-VHL) was cloned and its function was studied. It showed an overall 50% similarity to the human counterpart and 76% similarity in the crucial functional domain: the elongin C binding site. The putative d-VHL protein can bind Drosophila elongin C in vitro. During embryogenesis, d-VHL is expressed in the developing tracheal regions where tube outgrowth no longer occurs. Reduced d-VHL activity (using RNA interference methodology) caused breakage of the main vasculature accompanied by excessive looping of smaller branches, whereas over-expression caused a general lack of vasculature. Importantly, human VHL can induce the same gain-of-function phenotypes. VHL is likely involved in halting cell migration at the end of vascular tube outgrowth. Loss of VHL activity can therefore lead to disruption of major vasculature (as in the mouse embryo), which requires precise cell movement and tube fusion, or ectopic outgrowth from existing secondary vascular branches (as in the adult tumors) (Adryan, 2002)

Search PubMed for articles about Drosophila Vhl

Adryan B., et al. (2000). Tracheal development and the von Hippel-Lindau tumor suppressor homolog in Drosophila. Oncogene 19: 2803-2811. PubMed Citation: 10851083

Arquier, N., et al. (2006). Analysis of the hypoxia-sensing pathway in Drosophila melanogaster. Biochem. J. 393: 471-480. PubMed Citation: 16176182

Aso T., et al. (2000). Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity. Biochem. Biophys. Res. Commun. 276: 355-361. PubMed Citation: 11006129

Calzada M. J., et al. (2006). von Hippel-Lindau tumor suppressor protein regulates the assembly of intercellular junctions in renal cancer cells through hypoxia-inducible factor-independent mechanisms. Cancer Res. 66: 1553-1560. PubMed Citation: 16452212

Centanin, L., Ratcliffe, P. J. and Wappner, P. (2005). Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of Hypoxia-Inducible Factor-alpha/Sima. EMBO Rep. 6: 1070-1075. PubMed Citation: 16179946

Centanin, L., et al. (2008). Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting. Dev. Cell. 14: 547-558. PubMed Citation: 18410730

Champion, K. J., et al. (2008). Endothelial function of von Hippel-Lindau tumor suppressor gene: control of fibroblast growth factor receptor signaling. Cancer Res. 68: 4649-4657. PubMed Citation: 18559510

Chitalia, V. C., et al. (2008). Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL. Nat. Cell Biol. 10: 1208-1216. PubMed Citation: 18806787

Doronkin, S., et al. (2010). Dose-dependent modulation of HIF-1alpha/sima controls the rate of cell migration and invasion in Drosophila ovary border cells. Oncogene 29(8): 1123-34. PubMed Citation: 19966858

Duchi, S., et al. (2010). Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability. Development 137(9): 1493-503. PubMed Citation: 20388653

Frew I. J. and Krek W. (2007). Multitasking by pVHL in tumour suppression. Curr. Opin. Cell Biol. 19: 685-690. PubMed Citation: 18006292

Gorr, T. A., Gassmann, M. and Wappner, P. (2006). Sensing and responding to hypoxia via HIF in model invertebrates. J. Insect Physiol. 52: 349-364. PubMed Citation: 16500673

Harris T. J. and Peifer M. (2007). aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development. Dev. Cell 12: 727-738. PubMed Citation: 17488624

Harten S. K., et al. (2009). Regulation of renal epithelial tight junctions by the von Hippel-Lindau tumor suppressor gene involves occludin and claudin 1 and is independent of E-cadherin. Mol. Biol. Cell 20: 1089-1101. PubMed Citation: 19073886

Hergovich A., et al. (2003). Regulation of microtubule stability by the von Hippel-Lindau tumour suppressor protein pVHL. Nat. Cell Biol. 5: 64-70. PubMed Citation: 12510195

Hsu, T., et al. (2006). Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility. J. Biol. Chem. 281: 12069-12080. PubMed Citation: 16505488

Jarecki, J., Johnson, E. and Krasnow, M. A. (1999). Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF. Cell 99: 211-220. PubMed Citation: 10535739

Kaelin, W. G. (2005). The von Hippel-Lindau protein, HIF hydroxylation, and oxygen sensing. Biochem. Biophys. Res. Commun. 338: 627-638. PubMed Citation: 15952883

Kaelin W. G. (2008a). The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat. Rev. Cancer 8: 865-873. PubMed Citation: 18923434

Kaelin, W. G. and Ratcliffe, P. J. (2008b). Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol. Cell. 30: 393-402. PubMed Citation: 18498744

Kurihara, T., et al. (2010). von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina. Development 137(9): 1563-71. PubMed Citation: 20388654

Lolkema, M. P., et al. (2004). The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery. Exp. Cell Res. 301(2): 139-46. PubMed Citation: 15530850

Lolkema M. P., et al. (2007). The von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2. FEBS Lett. 581: 4571-4576. PubMed Citation: 17825299

Lolkema M. P., et al. (2008). Allele-specific regulation of primary cilia function by the von Hippel-Lindau tumor suppressor. Eur. J. Hum. Genet. 16: 73-78. PubMed Citation: 17912253

Lutz M. S. and Burk R. D. (2006). Primary cilium formation requires von hippel-lindau gene function in renal-derived cells. Cancer Res. 66: 6903-6907. PubMed Citation: 16849532

Mortimer, N. T. and Moberg, K. H. (2009). Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia. Dev. Biol. 329(2): 294-305. PubMed Citation: 19285057

Ohh, M., et al. (1998). The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol. Cell 1: 959-968. PubMed Citation: 9651579

Roe, J. S. and Youn, H. D. (2006). The positive regulation of p53 by the tumor suppressor VHL. Cell Cycle 5: 2054-2056. PubMed Citation: 16969113

Schermer, B., et al. (2006). The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth. J. Cell Biol. 175: 547-554. PubMed Citation: 17101696

Thoma, C. R., et al. (2009). VHL loss causes spindle misorientation and chromosome instability. Nat. Cell Biol. 11: 994-1001. PubMed Citation: 19620968

van Rooijen, E., et al. (2010). von Hippel-Lindau tumor suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish. Dis. Model Mech. 3(5-6): 343-53. PubMed Citation: 20335444

Yang, H., et al. (2007). pVHL acts as an adaptor to promote the inhibitory phosphorylation of the NF-kappaB agonist Card9 by CK2. Mol. Cell 28: 15-27. PubMed Citation: 17936701

date revised: 20 April 2011

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