A fish model for VHL disease
Von Hippel-Lindau (VHL) disease patients possess one defective copy of the VHL gene. Most VHL patients develop kidney tumors and this is a leading cause of death. The tumors are the result of accidental loss of the last remaining copy of the gene. In addition, many sporadic kidney tumors also lack VHL, underlining its general importance in tumor prevention. VHL functions in an "alarm system" that senses low oxygen levels, and loss of VHL protein leads to "false alarm".
This is not the only function of VHL, however, and it is unclear if this causes cancers. We have identified a VHL-like gene in zebrafish and inactivated it. Vhl deficient embryos show defects, suggesting that they try to adapt to low levels of oxygen. We aim to develop these embryos as a model for the human disease, particularly a model for kidney cancer.
Increased proliferation leads to increased PCNA staining in Hip mutants.
Hedgehog signalling and cell division
Hedgehog signalling is one of the few signalling pathways that regulates the development, differentiation and growth of a wide variety of tissues. The pathway is evolutionary ancient and active from fruit flyto man. Originally Hedgehog signalling was shown to be involved in patterning during embryonic development. However, it also regulates cell division in certain contexts. This has medical relevance because mutations in negative regulators of the pathway, such as PATCHED1 and SUPPRESSOR-OF-FUSED, are linked to cancer. In fact, the most frequent human cancer, basal cell carcinoma is caused by loss of the human PATCHED1. We are using zebrafish to study how the hedgehog signalling pathway can have two different outputs; patterning or proliferation. We exploit the fact that -uniquely in the zebrafish- there appear to be two patched genes that have different effects. Loss of one results mainly in patterning defects, whereas the other mainly results in increased cell division in the embryo.
Characterisation of a von Hippel Lindau 1 (vhl1) mutant in zebrafish, towards a model for kidney cancer
The VHL tumor suppressor gene functions in the oxygen-sensing pathway by causing degradation of hypoxia inducible factor (HIF) a under normoxic conditions. HIFa is the main regulator of the hypoxic response and therefore loss of VHL causes constitutive hypoxic signalling. Homozygous loss of VHL leads to kidney cancer both in sporadic cases and in VHL patients (via LOH).
We identified two zebrafish VHL-like genes, and created a knockout of the closest homolog (vhl1). Homozygous mutant embryos show a unique behavioral and morphological phenotype, which can be explained by abnormal hypoxic signalling, in addition to late kidney defects. This project aims to develop this vhl1 mutant as a model for VHL disease. Using genetic manipulations of relevant genes and we aim to understand the VHL "pathway" and generate a kidney tumor model in fish.
GFP hypoxia reporter embryos, exposed to normoxic (top) and hypoxic (bottom) conditions
Hedgehog signaling in patterning and growth
In a genetic "proliferation" screen with PCNA, we identified three genes that when mutated lead to a size increase in several tissues. Positional cloning of the mutated loci showed that they encode Patched2 (Ptc2) and two other negative regulators of the Hh pathway, Su(fu) and Hip. The growth defect of these mutants was unexpected, since previous publications describing artificial activation of this pathway did not report such effects.
Using TILLING we also identified a mutation in the zebrafish ptc1. Animals homozygous for this mutation show a spectrum of patterning defects similar to those induced by injection of hh mRNA and this phenotype is significantly enhanced in ptc1; ptc2 double mutants. It suggests that regulation of the Hh pathway by both ptc1 and ptc2 is critical for normal patterning of the embryo, whereas ptc2 is additionally required to modulate the control of growth by Hh signalling. Sequence comparison suggests that this subfunctionalisation may be unique to fish and we are currently doing experiments to understand and exploit this feature.
A,B) Wildtype and ptc1;ptc2 double mutant showing absence of eyes.
C,D) Abnormal somite patterning of ptc1;ptc2 double mutants leads to straight rather than V shaped myotome
Freek Van Eeden
MRC Centre for Developmental and Biomedical Genetics
The University of Sheffield
Firth Court, Western Bank
Sheffield S10 2TN
Room: D18 Firth Court
Office: +44 114 222 2348
Lab: +44 (0) 114 222 2381
Analysis of the role of VHL-like genes in DNA repair (link to BMS website).