Dr Christopher Wilkinson

Research interests

The role of centrosomes in early embryonic development in zebrafish

 

My interest lies in the roles of centrioles and associated proteins, as parts of the centrosome and cilium, in early vertebrate development, using zebrafish embryos as a model system. The centrosome is the microtubule-organising centre of the cell and so influences cell shape, polarity and migration. During cell division, the centrosome is duplicated and it forms part of the poles of the mitotic spindle that divides the duplicated chromosomes equally between daughter cells. The centrosome consists of two centrioles, cylindrical microtubule-based structures around which a complex of other proteins is formed (the pericentriolar matrix). The centriole is adapted in many cells to form the basal body from which cilia, hair-like, microtubule-based structures that protrude from the cell surface of many cell types.

Mutations in genes that encode components of the centrosome and cilium have been linked to a number of inherited, human developmental diseases: Bardet-Biedl, Alstrom, Joubert and oral-facial-digital syndromes - the 'ciliopathies'; a disease called primary microcephaly in which the size of the brain is reduced; and dwarfism.

My research is aimed at finding and characterising zebrafish embryos depleted of other centriolar proteins that give similar phenotypes in order to work out the developmental pathways in which these proteins and organelles are involved. I am particularly interested in proteins that contribute to the generation of cilia in early zebrafish embryos (red lines in Fig. 1A-C) and the control of cell division in the early embryonic brain (optical section showing nuclei in red and centrosomes in green, Fig. 1D). I use a variety of cell and molecular biology techniques in my research, including (confocal) (immuno)fluorscence microscopy and time-lapse imaging. I work with zebrafish embryos and zebrafish cell lines.

I have found two centrosome proteins whose depletion from zebrafish embryos gives a distinct phenotype: curved back and extra otoliths (bone-like structures) in the otic vesicle, the embryonic precursor to the ear. The embryos also display randomised left-right asymmetry so liver is on the left, heart on the right in half of the embryos. The cause of this phenotype in fact lies in cilia: they are much reduced in size in the developing fish embryo(Fig. 1 for cilia in different tissues of normal zebrafish embryos). In addition, cilia in the pronephros (embryonic kidney) are much shortened and the ducts of this organ are distended. This is reminiscent of zebrafish mutants for intraflagellar transport proteins, which move proteins up and down the cilium.

Future work will be to define how these two proteins contribute at the molecular level to construction of the cilium. I will also disrupt centrosome function by the depletion of specific centrosomal proteins from zebrafish embryos with the aim of determining the role of this organelle in brain development. Using a combination of live embryo imaging and immuno-histochemical analysis of tissue sections, I will assay the effect on the cell divisions of neural progenitors in the embryonic neuroepithelium.

Figure. Centrosomes and cilia in zebrafish embryos. A) cilia (red) in Küpffer's vesicle (involved in determining left/right asymmetry); B) cilia (red) and basal bodies (green) in the otic vesicle (develops into the fish ear); C) cilia and basal bodies in the pronephros (embryonic kidney); D) centrosomes (green, GFP-centrin) line the apical surface of the neuroepithelium in the diencephalon of the developing 48 h embryonic brain, nuclei labeled red (H2B-RFP). Compass shows orientation: D = dorsal, V = ventral, L = left, R = right.

ID: 10308