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von Hippel-Lindau disease
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Drosophila genes associated with von Hippel-Lindau disease
von Hippel-Lindau
Related terms

FGF pathway
Tracheal development
Relevant studies of von Hippel-Lindau disease

Shmueli, M.D., Schnaider, L., Herzog, G., Gazit, E. and Segal, D. (2014). Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome. PLoS One 9: e109864. PubMed ID: 25310726

The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches, this study demonstrates high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-α downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it (Shmueli, 2014).


  • Comparison of the human and Drosophila VHL proteins.
  • Folding and stability of dVHL are similar to pVHL.
  • Functional characterization of dVHL and pVHL.

The von Hippel-Lindau (VHL) syndrome is a rare hereditary cancer, associated with mutations in the VHL tumor suppressor gene. It is characterized by increased susceptibility to various tumors, both benign and malignant, including central nervous system haemangioblastomas, renal cysts and renal cell carcinoma (RCC) and phaeochromocytoma. The pVHL tumor suppressor protein is the substrate recognition subunit of a complex, comprising pVHL as well as Elongin C and B, termed VCB complex. This complex functions as part of an SCF-like ubiquitin-ligase that promotes the degradation of target proteins required for growth and vascularization of solid tumors. The pVHL protein has been implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase which targets the Hypoxia-Inducible Factor (HIF1-α) for proteasomal degradation. Upon decrease in oxygen levels residues P564 and/or P402, in the oxygen dependent degradation domain (ODD, residues 401–603) of HIF1-α, are hydroxylated and interact with Y98 in pVHL leading to VCB-mediated E3 degradation of HIF1-α. This hydroxylation reaction is mediated by the prolyl-hydroxylase domain protein (PHD). Under hypoxic conditions, the prolyl hydroxylase is inactive and the HIF1-α subunit is not hydroxylated. HIF1-α then dimerizes with the β subunit (HIF1-β), which is constitutively expressed. The HIF heterodimer translocates to the nucleus where it functions as a transcription factor. The best known target genes of HIF encode proteins involved in glycolysis, glucose transport (Glut-1), angiogenesis (vascular endothelial growth factor (VEGF)) and erythropoiesis (erythropoietin), i.e., proteins that mediate the cellular response and adaptation to hypoxic conditions. The VHL gene encodes two biologically active isoforms of pVHL (19 kDa and 30 kDa) as a result of in-frame alternative AUG codon usage. However, no functional significance has been assigned to the extra 53 amino acids at the N-terminus of the large isoform. pVHL shuttles back and forth between the nucleus and the cytoplasm. The short isoform of pVHL is localized primarily to the nucleus whereas the long isoform is more frequently associated tightly with microtubules in the cytoplasm (Shmueli, 2014).

Lower model organisms such as Drosophila melanogaster and Caenorhabditis elegans have often provided the first glimpse into the mechanism of action of human cancer-related proteins, thus making a substantial contribution to the elucidation of the molecular basis of the disease. More than 50% of the proteins that are associated with human diseases, including cancer, have orthologues in D. melanogaster. In many cases, the function of a given protein is also conserved evidenced by the fact that the corresponding human protein can rescue the loss of function of its Drosophila orthologue. Thus, studies of the fly orthologue as well as investigation of the transgenic human proteins in flies, which are highly amenable for genetic manipulations, provide important information on the human disease-associated protein and the pathways it is involved in. This is exemplified especially regarding to the contribution of Drosophila to basic and applied aspects of cancer research. The highly ordered, lattice-like, arrangement of the ommatidia in the fly eyes is especially suitable for large scale screens for genetic modifiers and for drug candidates. Flies have also greatly facilitated drug screening. Studies show that the Drosophila VHL (dVHL) can interact with Drosophila Elongin C in vitro and form a complex with the human Elongin CB complex and mouse Rbx-1, but not with the human Elongin A. Thus, the ubiquitin ligase function of pVHL is conserved in Drosophila. It is also known that dVHL is involved in degradation of hydroxylated oxygen-dependent degradation domain (ODD) of both the human HIF1-α or of the Drosophila homolog, SIMA. Studies of dVHL have suggested the involvement of the VHL protein in regulation of endocytosis and epithelial cell motility which was subsequently demonstrated for pVHL as well. Research on dVHL has also indicated a role for the VHL protein in microtubule stabilization during epithelial morphogenesis; yet, this remains to be demonstrated for pVHL as well (Shmueli, 2014).

The results presented in this study regarding the structural similarity between the dVHL and pVHL proteins provide insights into their functional homology. It is known that pVHL is a molten globule protein that it is intrinsically disordered but is functional as such. This study shows that dVHL, like pVHL, has a secondary structure but lacks well-defined tertiary structure and attains a molten globule conformation. Also, dVHL, like pVHL, is functional without being uniquely folded into a rigid 3D structure; thus, dVHL, like pVHL, is an intrinsically disordered protein (IDP). The demonstration of homology between IDPs is a major endeavor since they do not display sequence conservation. In addition, due to the lack of well-defined 3D structure, IDP regions fail to scatter X-rays coherently, hampering attempts to solve their structure by X-ray crystallography (Shmueli, 2014).

The in silico predicted homology in the conformation of the key functional amino acids of dVHL and pVHL, as well as their evolutionary conservation, supported by in vitro results in this study, provide a structural basis for understanding the homology in their function as well as their ability to interact with each other’s binding partners. dVHL can bind in vivo the hydroxylated ODD domain of the human HIF1-α and target it for degradation and it was shown that transgenic pVHL can do the same in flies. Moreover, it was demonstrated that transgenic pVHL can rescue the lethal phenotype of dVHL null mutants of Drosophila. These results suggest that pVHL is capable of interacting, in flies, with the Drosophila members of the VCB complex, thus regulating the fly HIF1-α homolog, SIMA. Indeed, this rescue involves regulation of the HIF1-α/SIMA dependent pathway, evidenced by the level of its target genes (Shmueli, 2014).

The Drosophila model described in this study should facilitate further studies on the function of pVHL and the pathways it regulates. The rough eye phenotype caused by over-expression of SIMA, which was shown to be suppressed by transgenic expression of normal pVHL, should be highly conducive for conducting screens for drug candidates for the VHL syndrome in Drosophila whole model organism. These, can be subsequently verified molecularly by examining the downstream HIF1-α dependent pathway which are conserved between flies and humans (Shmueli, 2014).

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Shmueli, M. D., Levy-Kanfo, L., Haj, E., Schoenfeld, A. R., Gazit, E. and Segal, D. (2018). Arginine refolds, stabilizes, and restores function of mutant pVHL proteins in animal model of the VHL cancer syndrome. Oncogene. PubMed ID: 30194449


Shmueli, M. D., Levy-Kanfo, L., Haj, E., Schoenfeld, A. R., Gazit, E. and Segal, D. (2018). Arginine refolds, stabilizes, and restores function of mutant pVHL proteins in animal model of the VHL cancer syndrome. Oncogene. PubMed ID: 30194449

Arginine refolds, stabilizes, and restores function of mutant pVHL proteins in animal model of the VHL cancer syndrome

The von Hippel-Lindau (VHL) syndrome is a rare inherited cancer, caused by mutations in the VHL gene, many of which render the VHL protein (pVHL) unstable. pVHL is a tumor-suppressor protein implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase complex which targets the hypoxia-inducible factor (HIF) for degradation. Previous analyses have shown that common oncogenic VHL mutations render pVHL less stable than the wild-type protein, distort its core domain and as a result reduce the ability of the protein to bind its target HIF-1alpha. Among various chemical chaperones tested, arginine was the most effective in refolding mutant of pVHL. This study examined the consequences of administering L- or D-arginine to a Drosophila VHL model and to human renal carcinoma cells, both expressing misfolded versions of human pVHL. Arginine treatment increased pVHL solubility in both models and increased the half-life of the mutant pVHL proteins in the cell culture. In both models, L- as well as D-arginine enhanced the ability of wild-type pVHL and certain misfolded mutant versions of pVHL to bind ODD, the HIF-derived target peptide, reflecting restoration of pVHL function. Moreover, continuous feeding of Drosophila expressing misfolded versions of pVHL either L- or D-arginine rich diet rescued their lethal phenotype. Collectively, these in vivo results suggest that arginine supplementation should be examined as a potential novel treatment for VHL cancer syndrome (Shmueli, 2018).

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Delgehyr, N., Wieland, U., Rangone, H., Pinson, X., Mao, G., Dzhindzhev, N.S., McLean, D., Riparbelli, M.G., Llamazares, S., Callaini, G., Gonzalez, C. and Glover, D.M. (2012). Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability. Proc Natl Acad Sci U S A 109: 5729-5734. PubMed ID: 22451918

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. This study shows that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting β-tubulin, suggesting Mgr function is required for tubulin stability. Instability of β-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic β-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading to a proposed requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not (Delgehyr, 2012).


  • Mgr is a subunit of the highly conserved Gim complex/Prefoldin.
  • Spindle abnormalities result from tubulin destabilization following Mgr depletion.
  • Levels of free αβ-tubulin sensitize Mgr activity.
  • Mgr and Vhl cooperate to control the degradation of αβ-tubulins.

The finding that the Drosophila merry-go-round gene encodes a subunit of the Prefoldin complex accounts for aberrant structure and function of spindles and centrosomes in cells depleted of its gene product. The inability to correctly fold tubulins in Prefoldin-deficient cells leads to tubulin instability and, hence, defects that can be phenocopied by depleting β- or γ-tubulin. However, whereas β-tubulin depletion phenocopied all of the defects observed, γ-tubulin depletion only recapitulates some of them. The more dramatic phenotypes seen in Mgr-deficient cells expressing high levels of tubulin (primary spermatocytes and neuroblasts expressing a β-tubulin transgene) suggest that the Prefoldin complex is critical to maintain tubulin levels above a certain threshold of tubulin expression. This finding could be a consequence of the impact of an excess of tubulin upon its complex folding pathway. Interestingly, in mammalian cells, increased soluble tubulin, in response to a MT-destabilizing agent, leads to the rapid degradation of tubulin. In Drosophila, tubulins in the testes are the most affected by the absence of Mgr compared with other tissues. Indeed, it may be of particular importance to regulate tubulin levels at the late stages of spermatogenesis, where the very large meiotic cells are provided with proportionally large amounts of tubulin that are used in the meiotic spindle but have a major additional purpose: the building of the sperm tail. Similarly, in the mouse, the effects of depletion or mutation of prefoldin subunits are largely restricted to the brain, where tubulin levels are also very high. Whether this tissue specificity is a consequence of tubulin levels will be an interesting question to address. Finally, the demonstration that Vhl is required for tubulin destruction in the absence of Mgr and the ability of Vhl to interact with tubulin monomers and dimers raises the possibility that its role as an E3 ubiquitin-protein ligase could come to play in regulating tubulin levels (Delgehyr, 2012).

The idea that Vhl and Prefoldin can cooperate in regulating protein stability has been raised earlier by another study that identified the prefoldin subunit VBP1 as a binding partner of the HIV-1 viral integrase and suggested it to mediate the interaction of the integrase with the Cul2-Vhl E3-Ubiquitin ligase. This finding led to the suggestion for a role for prefoldin at a pivotal part of the pathway that would determine whether a protein was passed on to the CCT chaperonin for folding or to the proteasome for degradation. Similarly, this study speculates that prefoldin as a partner of Vhl may well serve a key role in regulating the equilibrium between tubulin targeted for destruction or for folding and incorporation into MTs. The concentration of assembly-competent tubulin must be tightly controlled because it affects cytoskeletal dynamics. Vhl might contribute to this influence by an effect on MT dynamics through interaction with MAPs on the MT lattice and by intervening in the regulation of tubulin folding. There is growing evidence for a critical function of Vhl in stabilizing cytoplasmic MTs and axonemal MTs in response to levels of soluble tubulin. Reciprocally, MT stability can contribute to regulating levels of proteins that are targets of the Cul2-Vhl E3-Ubiquitin ligase, such as the HIF proteins, the levels of which fall when their mRNAs accumulate in cytoplasmic P-bodies for translational repression following MT disruption. It will be important in future to consider the roles played by the Prefoldin complex and Vhl to understand the interrelationships between the machinery regulating tubulin levels in relation to MT stability, both in normal and tumor cells (Delgehyr, 2012).

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Hsouna, A., Nallamothu, G., Kose, N., Guinea, M., Dammai, V. and Hsu, T. (2010). Drosophila von Hippel-Lindau tumor suppressor gene function in epithelial tubule morphogenesis. Mol Cell Biol 30: 3779-3794. PubMed ID: 20516215

Mutations in the human von Hippel-Lindau (VHL) gene are the cause of VHL disease that 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-α). However, many HIF-independent functions of VHL have been identified. Earlier studies also indicate that the canonical function cannot fully explain the VHL mutant cell phenotypes, although it is still unclear how many of these noncanonical functions relate to the pathophysiological processes because of a lack of tractable genetic systems. This study reports the first genomic mutant phenotype of Drosophila melanogaster VHL (dVHL) in the epithelial tubule network, the trachea, and shows that dVHL regulates branch migration and lumen formation via its endocytic function. The endocytic function regulates the surface level of the chemotactic signaling receptor Breathless (Btl) and promotes clearing of the lumen matrix during maturation of the tracheal tubes. Importantly, the regulatory function in tubular morphogenesis is conserved in the mammalian system, as conditional knockout of Vhl in mouse kidney also results in similar cell motility and lumen phenotypes (Hsouna, 2010).


  • Characterization of the dVHL1 allele.
  • The dVHL loss-of-function phenotype results in abnormal tracheal structure during embryonic development.
  • dVHL1 germ line mutants show complete failure in forming trachea.
  • dVHL mutant tracheal cells exhibit ectopic migration phenotypes.
  • dVHL mutants display overactive FGFR/Btl signaling.
  • dVHL protein interacts with abnormal wing discs (awd).
  • Vhl knockout in mouse kidney tubules generates lumen and branching defects.

This study shows that dVHL regulates tracheal tubule development in two aspects—branch migration and lumen formation. These two aspects of tracheal morphogenesis are regulated by the same endocytic function; one involves internalization of the Btl signaling receptor, and the other involves resorption of lumen materials (Hsouna, 2010).

The study also shows that dVHL genetically interacts with endocytic pathway components such as awd, shibire (shi) and Rab5 in both branch migration and lumen formation phenotypes but interacts with btl signaling pathway components only in branch migration phenotypes. The lumen defects in the dVHL mutant are similar to those found in the wurst mutant. wurst encodes a transmembrane protein that promotes clathrin-mediated endocytosis of lumen material. wurst function is specific for lumen maturation and, unlike dVHL, has little effect on branch migration. This indicates that wurst may be specific for pinocytosis (uptake of extracellular materials) and not involved in internalization of surface proteins, while the dVHL- and awd-regulated dynamin-Rab5 pathway is necessary for both (Hsouna, 2010).

It was earlier reported that some of the tracheal tubule migration defects generated by dVHL RNA interference could be attributed to upregulation of Sima, the Drosophila homolog of HIF-α, which in turn upregulates btl transcription. Hypoxia-induced, Sima-dependent btl transcription has also been demonstrated in terminal branching in the larval trachea. It is possible that the endocytic and the HIF-dependent functions of VHL are not mutually exclusive. On the other hand, it has been shown that the stereotypic tracheal branch migration pattern in the embryo (as opposed to the terminal branching in larvae) is normally not dependent on sima function. Also, overexpression of the wild-type btl transgene in the embryonic trachea using the cognate btl promoter, although exhibiting ectopic elevation of the btl transcript level, could not lead to embryonic tracheal defects. This indicates that posttranscriptional regulation is the major mechanism for modulating active Btl level and thus Btl signaling. Indeed, it was shown in this study that the exogenously expressed Btl::GFP protein is under stringent control at the protein level. Also, sima can only modestly suppress the dVHL1 branch migration phenotype. It is interesting to point out that a robust rescue of phenotypes in the btl promoter-driven dVHL RNA duplex by sima heterozygotes has been shown earlier. This may be because the exogenous btl promoter, presumably positively regulated by sima, is itself downregulated in the sima mutant, thereby diminishing the dVHL duplex expression, resulting in apparent rescue (Hsouna, 2010).

It should be noted that primary and secondary tracheal tubule development during embryogenesis is a stereotypic, genetically programmed process. Tissue microenvironmental factors such as hypoxia do not play a significant role in this process. On the other hand, in late embryonic and larval stages, when the trachea system extends to connect with internal organs, localized hypoxia in the target tissue is critical in inducing FGF/bnl expression and thus promoting terminal branching. HIF-α/Sima has been shown to also play an important role in the terminal tracheal tubule cells in sensing hypoxia and inducing FGFR/btl expression. This mechanism, in the absence of dVHL mutation, can contribute to tracheal branching probably because it is sensitized by the overproduced Bnl ligand in hypoxic conditions. It is conceivable that dVHL loss of function during embryogenesis can result in similar upregulation of the btl gene transcription. However, results in this study indicate that in nonhypoxic development of trachea in the embryonic stage, the additional function of dVHL in regulated protein internalization plays the major role in tracheal morphogenesis (Hsouna, 2010).

Most interestingly, the study demonstrates that the ectopic branching of the tubule epithelial cells and the malformation of lumen phenotypes are reproducible in the kidney tubules of mice with conditional Vhl knockout and in organoid culture using primary tubule cells. This is highly significant, since dilated tubules (minicysts) have been documented as preceding renal cell carcinoma. Thus, these findings provide a plausible mechanistic explanation, involving increased cell motility and disruption of tubule epithelium, for the etiology of VHL mutant kidney cancer. In addition, another study implicated dVHL in the morphogenesis of organ-associated epithelium, the follicle cells in the ovary. This function is mediated by another HIF-independent activity of dVHL that stabilizes microtubule bundles. Future studies should exploit further the Drosophila genetic system for elucidating how various VHL functions and a myriad of disease-related VHL mutations may differentially affect the pathophysiological roles of this interesting tumor suppressor gene (Hsouna, 2010).

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Duchi, S., Fagnocchi, L., Cavaliere, V., Hsouna, A., Gargiulo, G. and Hsu, T. (2010). Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability. Development 137: 1493-1503. PubMed ID: 20388653

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 there is evidence 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. These results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity (Duchi, 2010).


  • Drosophila VHL mutant follicle cells exhibit epithelial defects.
  • Altered epithelial marker expression in VHL mutant cells.
  • Homozygous VHL1 egg chambers show severe epithelial defects.
  • VHL functions via stabilizing microtubules (MTs).
  • VHL regulates MTs and aPKC stability.
  • A disease-related VHL mutant defective in MT stabilization cannot rescue the aPKC localization phenotype.

This study shows that Drosophila VHL is important for establishing and maintaining epithelial integrity via its regulation of microtubule (MT) and aPKC stability. MT disruption and epithelial phenotypes early in oogenesis were observed. 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. It was shown that disrupted MTs interfere with proper localization of aPKC, which in turn leads to mislocalization of downstream epithelial markers and epithelial defects. 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. Earler studies using RNA interference-mediated knockdown demonstrate a morphogenic role of VHL in trachea development. 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. This study favors a separate, tissue-specific function for VHL as 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. 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. The study therefore concludes 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, 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, 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. Future studies using the epithelial system should also address this issue in vivo. Also interestingly, it was shown that the VHLYH mutant (single amino acid substitution mutation of tyrosine to histidine at position 51, equivalent to human Y98) can associate with MT but has little MT-stabilizing activity. This suggests that the VHLYH 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 (generated by replacing the wild-type copy, via homologous recombination, with a deletion that removes 81 codons encompassing the first two in-frame AUGs). It has been shown that tracheal defects are the major embryonic phenotype observed in VHL mutant. In the course of attempting to rescue the tracheal phenotype with btl-driven VHL, rescued homozygous adults are present. 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, there is also evidence that VHL is a multifunctional protein. It can function as a regulator of matrix deposition, integrin assembly, endocytosis, kinase activity, senescence, protein stabilities and tight junction formation, among many others. 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. 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. 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).

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Doronkin, S., Djagaeva, I., Nagle, M.E., Reiter, L.T. and Seagroves, T.N. (2010). Dose-dependent modulation of HIF-1alpha/sima controls the rate of cell migration and invasion in Drosophila ovary border cells. Oncogene 29: 1123-1134. PubMed ID: 19966858

This study analyses the role of the hypoxic response during metastasis in migrating border cells of the Drosophila ovary. Acute exposure to 1% O2 delays or blocks border cell migration (BCM), whereas prolonged exposure results in the first documented accelerated BCM phenotype. Similarly, manipulating the expression levels of sima, the Drosophila hypoxia-inducible factor (HIF)-1α ortholog, reveals that Sima can either block or restore BCM in a dose-dependent manner. In contrast, over-expression of Vhl (Drosophila von Hippel–Lindau) generats a range of phenotypes, including blocked, delayed and accelerated BCM, whereas over-expression of hph (Drosophila HIF prolyl hydroxylase) only accelerates BCM. Mosaic clone analysis of sima or tango (HIF-1β ortholog) mutants reveals that cells lacking Hif-1 transcriptional activity are 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 is 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).


  • Vhl/VHL over-expression causes pleiotropic defects.
  • DE-cadherin levels are modulated by Vhl and sima.

In addition to other investigations, this study also investigated whether increasing Vhl expression, which would also be expected to decrease accumulation of Sima, would affect BCM. Stocks were created in which expression of full-length, FLAG epitope-tagged Vhl or human VHL was driven in border cells. A broad spectrum of phenotypes was observed ranging from normal to accelerated to blocked migration using either driver. Compared with wild-type chambers dissected at stage 10, in which migration was complete, up to 40% of egg chambers show a delay in migration. However, up to 10% of chambers exhibit accelerated migration, moving beyond the border between the nurse cells and the oocyte. Although 50–60% of UAS–Vhl egg chambers display normal BCM, a small fraction (7%) show complete migration failure. Similar results were obtained for the c522–GAL4 driver at stages 9 and 10 (Doronkin, 2010).

It is noteworthy that ectopic expression of human VHL in the Drosophila ovary using either the slbo-GAL4 or c522-GAL4 driver also produces a similar range of phenotypes. VHL over-expression causes delays in migration of 39/29% of chambers, blocks migration in 7/6% of chambers, and accelerates migration in 5/8% of egg chambers, respectively. Together, these data demonstrate that BCM is more sensitive to ectopic expression of Sima than Vhl and provide additional evidence that the VHL pathway is highly conserved in Drosophila. Finally, expression of either Vhl or Hph increases BCM without damaging the cortical cytoskeleton. In a slbo–GAL4>UAS–Vhl egg chamber exhibiting the accelerated phenotype, the abnormal, posterior location of accelerated clusters is consistently accompanied by a stretching, but not complete disruption, of the oocyte/nurse cell actin network (Doronkin, 2010).

One of the key downstream effector molecules in collective BCM is Drosophila E-cadherin (DE-cadherin; encoded by shotgun). The VHL/HIF axis has been shown to regulate E-cadherin levels in human renal cancer cells. This study compared DE-cadherin expression levels by immunostaining fixed egg chambers dissected from UAS–sima, UAS–Vhl or sima mosaic clone border cell clusters. In wild-type clusters, DE-cadherin accumulates to the highest levels at the interface between the border cells and the polar cells, located at the center of the cluster. Accumulation is less pronounced between the cluster and the nurse cells. In UAS–sima clusters, expression of DE-cadherin is enhanced compared with control clusters at the boundaries between individual border cells. Similarly, in delayed UAS–Vhl clusters, DE-cadherin appears to more strongly accumulate at the interface of the cluster and the surrounding nurse cells rather than between the border cells and polar cells. Finally, in all accelerated UAS–Vhl clusters, DE-cadherin expression is potently reduced. It is possible that increased accumulation of DE-cadherin in the border cell clusters in response to Sima over-accumulation may have strengthened cell–cell adhesions leading to stalled migration. Similarly, reduction of the DE-cadherin levels in sima-mutant cells could have produced weaker contacts between the neighboring nurse cells and the migrating border cells, which would explain the accelerated cell migration observed in UAS–Vhl expressing cells (Doronkin, 2010).

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

The tracheal system of Drosophila melanogaster 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 identifies two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes: 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, 2009).


  • Stage-specific effects of hypoxia on embryonic tracheogenesis.
  • dVHL is required to suppress the tracheal hypoxic response.
  • dVHL genetically antagonizes sima in the embryonic trachea.
  • dVHL suppresses btl expression in the embryo.
  • dVHL and ago synergize to control embryonic tracheogenesis

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 examines 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, 2009).

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, by the modest ability of trh alleles to suppress dVHLi phenotypes, and by the 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, 2009).

This study 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, 2009).

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. 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, 2009).

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 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. 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. Whether this type of mechanism operates in embryos, or whether embryos ever experience localized hypoxia in non-tracheal cells, has not been established (Mortimer, 2009).

Data from this study also 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 disrupts normal tracheogenesis, but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. This study shows 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. 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 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, while expression of a constitutively active btl chimera (btlλ) 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, 2009).

In addition to sima and Btl/FGF pathway mutants, dVHL also shows very strong genetic interactions with alleles of the ago ubiquitin ligase subunit. The interactions are consistent with the ability of ago to modulate hypoxia sensitivity in the embryo, and suggest a speculative model in which each ligase acts through its own target — Sima or Trh — to regulate btl transcription in tracheal cells. Given that the human orthologs of dVHL and ago are significant tumor suppressor genes, it is intriguing to consider whether their ability to co-regulate tubular morphogenesis in the Drosophila embryo is conserved in mammalian development and disease (Mortimer, 2009).

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Aso, T., Yamazaki, K., Aigaki, T. and Kitajima, S. (2000). Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity. Biochem Biophys Res Commun 276: 355-361. PubMed ID: 11006129

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 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 show 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, hypoxia-inducible factor (HIF)-1α is the ubiquitination target of both human and Drosophila VHL complexes (Aso, 2000).

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Hsu, T. (2012). Complex cellular functions of the von Hippel-Lindau tumor suppressor gene: insights from model organisms. Oncogene 31: 2247-2257. PubMed ID: 21996733

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von Hippel-Lindau tumor suppressor protein, a component of an E3 ubiquitin ligase complex, is required for degradation of HIF-1

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Date revised: 20 June 2015

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