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Using mouse models to investigate the roles of EVC and EVC2 in cilia and development

Reference	BB/D004926/1
Principal Investigator / Supervisor	Professor Judith Goodship
Co-Investigators / Co-Supervisors	Dr Helen Blair
Institution	Newcastle University
Department	Institute of Human Genetics
Funding type	Research
Value (£)	319,486
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	30/04/2009
Duration	43 months

Abstract

Through mapping a human disorder, Ellis-van Creveld syndrome (EvC), we have identified two genes (EVC and EVC2) that play a role in skeletal development. We have shown that Evc is present at the base of the cilia and, in serum starved human cells, within the nucleus. The function of primary cilia in chondrocytes and osteocytes is unknown. As many primary cilia have a sensory role it is possible that it Evc is involved in relaying signals to the nucleus. We plan to use electron microscopy to refine the localisation, which could be pericentriolar material, distal or subdistal centriolar appendages or transition zone, at the base of the cilium. We will also image these structures in cells lacking Evc/ Evc2. We plan to assess whether Evc and Evc2 basal body localisation is microtubule dependent (by nocodoazole treatment) and dynein-dynactin motor dependent (by overexpression of a p50/dynamitin construct). We will add inhibitors of protein translation (cyclohexamide) prior to serum starving to determine if the nuclear localisation under these conditions results from translocation or de novo protein synthesis. Preliminary examination of an Evc targetted knockout we have made indicates that the mouse phenotype replicates Ellis-van Creveld syndrome. We have made constructs to target Evc2 and to target both genes (as the genes are only 1.6 kb apart this could not be achieved by crosses between the Evc and Evc2 targetted mice). We will phenotype Evc-/-, Evc2-/-, Evc+/-Evc2+/- and Evc-/-Evc2-/- mice using standard histology techniques and alizarin red/alcian blue staining. We will study the lacZ (Evc) and EGFP (Evc2) expression patterns in heterozygotes. The temperospatial expression pattern and the first histological difference detected between mutant with wild type littermates will be the basis for selection of relevant markers for RNA TISH/ antibody staining.

Summary

Ellis-van Creveld syndrome is named after the two doctors who first described the condition. People with this condition have short ribs, short limbs, and abnormalities of their teeth and nails. We mapped this condition and went on to find two genes that, when faulty, cause the condition (EVC and EVC2). The two genes are very close together and it seems likely they are regulated from the short bit of DNA between them because they are transcribed (read)in opposite directions from this region. We have made mice lacking evc and these mice have the same features as the patients indicating that they are a good model to work out when and where the first abnormalities occur in the developing skeleton. This in turn will give us information about normal skeletal development. We will study the appearance of the developing bones in the mice lacking the protein. We are also making a mouse that lacks Evc2. In making these mice we have included tags that will be expressed where the protein would normally be expressed and which can easily be visualised. We will use these tags to study where and when the proteins would be expressed. We also want to know exactly where the proteins are in cells. The experiments we have done so far have given fascinating results. Many cells in the body have a hair like projection called a cilium. These were first seen when scanning electron microscopy was introduced thirty years ago but no-one knew what they did. Evc and Evc2 localise to the base of the cilia. This is the first clue that these cilia have a role in skeletal development. We will use electron microscopy to look at this in even more detail. We will also use electron microscopy to look at this region of cells in the mice lacking the proteins; we have already shown that these cells make cilia but electron microscopy may pick up a difference in their structure. We will also study how Evc and Evc2 get to the base of cilia. It is likely that they are carried along microtubules by a molecular motor called dynein and we can test this using chemicals that disrupt tubules and the dynein motor respectively. We have also seen Evc in the nucleus of the cell which is where genes are regulated. It is possible that cilia are like antennae sensing things outside the cell, Evc and Evc2 could be involved in relaying such signals to the nucleus which in turn will switch genes on/off leading to a response. We hope that through this project we will learn more about primary cilia and skeletal development.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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