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Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos.

Wilkinson CJ, Carl M, Harris WA - BMC Cell Biol. (2009)

Bottom Line: Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase) interacting domain.Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened.We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK. Christopher.Wilkinson@rhul.ac.uk

ABSTRACT

Background: The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms.

Results: We describe zebrafish (Danio rerio) embryos depleted for two uncharacterised, centrosome proteins, Cep70 and Cep131. The phenotype of these embryos resembles that of zebrafish mutants for intraflagellar transport proteins (IFTs), with kidney and ear development affected and left-right asymmetry randomised. These organs and processes are those affected in Bardet-Biedl syndrome and other similar diseases. Like these diseases, the root cause of the phenotype lies, in fact, in dysfunctional cilia, which are shortened but not eliminated in several tissues in the morphants. Centrosomes and basal bodies, on the other hand, are present. Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase) interacting domain. However, we could not detect in yeast two-hybrid assays any interaction with the deacetylase that controls cilium length, HDAC6, or any of the IFTs that we tested.

Conclusion: Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened. We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

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Confocal wholemount (immuno)fluorescence localisation of Cep70 in zebrafish embryos. (A and E) In embryos injected with GFP-Cep70 and H2B-RFP, the Cep70 signal, though weak, is observed at the apical surface of cells in the otic vesicle (A) and eye (E) at 24 h.p.f. . Long dashed lines indicate the apical surface: interior to the circle in (A) and above the line in (E). Other panels: 24 h.p.f. zebrafish embryos were probed with anti-Cep70 (green) and either anti-acetylated or anti-gamma tubulin (red). (B, C, D) Otic vesicle stained for gamma tubulin (C), Cep70 (D) and merged picture (B) showing that the two signals coincide. Arrowheads point to the non-specific staining of the otoliths. (F) and (G) show the eye and otic vesicle; Cep70 can be seen at the base of cilia stained for acetylated-tubulin. (H) shows the region around one of the clusters of tether cells in the otic vesicle where the position of Cep70 can be seen more clearly; an inset shows one basal body and cilium. Yellow colour, the overlap of Cep70 and acetylated-tubulin signal is due to the angle at which some of the cilia are seen. Again, a long dashed line indicates the apical surface of the eye in (F). Short dashed lines delineate the otic vesicle in (B, C, D and G). (I) and (J) show Cep70 and cilia in the pronephros and spine canal. Again the Cep70 can be seen at the base of cilia. Orientation of the specimens with regard to the embryo is shown by the compass in the panel: A = anterior, P = posterior, D = dorsal, V = ventral. Scale bar: 20 μm.
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Figure 3: Confocal wholemount (immuno)fluorescence localisation of Cep70 in zebrafish embryos. (A and E) In embryos injected with GFP-Cep70 and H2B-RFP, the Cep70 signal, though weak, is observed at the apical surface of cells in the otic vesicle (A) and eye (E) at 24 h.p.f. . Long dashed lines indicate the apical surface: interior to the circle in (A) and above the line in (E). Other panels: 24 h.p.f. zebrafish embryos were probed with anti-Cep70 (green) and either anti-acetylated or anti-gamma tubulin (red). (B, C, D) Otic vesicle stained for gamma tubulin (C), Cep70 (D) and merged picture (B) showing that the two signals coincide. Arrowheads point to the non-specific staining of the otoliths. (F) and (G) show the eye and otic vesicle; Cep70 can be seen at the base of cilia stained for acetylated-tubulin. (H) shows the region around one of the clusters of tether cells in the otic vesicle where the position of Cep70 can be seen more clearly; an inset shows one basal body and cilium. Yellow colour, the overlap of Cep70 and acetylated-tubulin signal is due to the angle at which some of the cilia are seen. Again, a long dashed line indicates the apical surface of the eye in (F). Short dashed lines delineate the otic vesicle in (B, C, D and G). (I) and (J) show Cep70 and cilia in the pronephros and spine canal. Again the Cep70 can be seen at the base of cilia. Orientation of the specimens with regard to the embryo is shown by the compass in the panel: A = anterior, P = posterior, D = dorsal, V = ventral. Scale bar: 20 μm.

Mentions: In 24 h.p.f. embryos, GFP-Cep70 localises to the apical surface of cells in the otic vesicle (Fig. 3A) and eye (Fig. 3E) with a punctate appearance. This can be replicated with wholemount immunofluorescence staining of 24 h.p.f. embryos with rabbit anti-Cep70 485-4 (Fig. 3F, G). This staining coincides with that of mouse monoclonal GTU-88 to gamma tubulin which labels centrosomes and basal bodies (Fig. 3B – merge of C and D) and lies at the base of cilia labelled with mouse monoclonal 6-11B-1 to acetylated tubulin (Fig. 3G), shown in detail in Fig. 3H and inset. In the otic vesicle there is non-specific staining of the otoliths but in other ciliated tissues, only the basal bodies are labelled (Fig. 3F (eye), I (pronephros), J (spine canal)). In ciliated cells, therefore, Cep70 localises to the basal bodies of cilia.


Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos.

Wilkinson CJ, Carl M, Harris WA - BMC Cell Biol. (2009)

Confocal wholemount (immuno)fluorescence localisation of Cep70 in zebrafish embryos. (A and E) In embryos injected with GFP-Cep70 and H2B-RFP, the Cep70 signal, though weak, is observed at the apical surface of cells in the otic vesicle (A) and eye (E) at 24 h.p.f. . Long dashed lines indicate the apical surface: interior to the circle in (A) and above the line in (E). Other panels: 24 h.p.f. zebrafish embryos were probed with anti-Cep70 (green) and either anti-acetylated or anti-gamma tubulin (red). (B, C, D) Otic vesicle stained for gamma tubulin (C), Cep70 (D) and merged picture (B) showing that the two signals coincide. Arrowheads point to the non-specific staining of the otoliths. (F) and (G) show the eye and otic vesicle; Cep70 can be seen at the base of cilia stained for acetylated-tubulin. (H) shows the region around one of the clusters of tether cells in the otic vesicle where the position of Cep70 can be seen more clearly; an inset shows one basal body and cilium. Yellow colour, the overlap of Cep70 and acetylated-tubulin signal is due to the angle at which some of the cilia are seen. Again, a long dashed line indicates the apical surface of the eye in (F). Short dashed lines delineate the otic vesicle in (B, C, D and G). (I) and (J) show Cep70 and cilia in the pronephros and spine canal. Again the Cep70 can be seen at the base of cilia. Orientation of the specimens with regard to the embryo is shown by the compass in the panel: A = anterior, P = posterior, D = dorsal, V = ventral. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2662791&req=5

Figure 3: Confocal wholemount (immuno)fluorescence localisation of Cep70 in zebrafish embryos. (A and E) In embryos injected with GFP-Cep70 and H2B-RFP, the Cep70 signal, though weak, is observed at the apical surface of cells in the otic vesicle (A) and eye (E) at 24 h.p.f. . Long dashed lines indicate the apical surface: interior to the circle in (A) and above the line in (E). Other panels: 24 h.p.f. zebrafish embryos were probed with anti-Cep70 (green) and either anti-acetylated or anti-gamma tubulin (red). (B, C, D) Otic vesicle stained for gamma tubulin (C), Cep70 (D) and merged picture (B) showing that the two signals coincide. Arrowheads point to the non-specific staining of the otoliths. (F) and (G) show the eye and otic vesicle; Cep70 can be seen at the base of cilia stained for acetylated-tubulin. (H) shows the region around one of the clusters of tether cells in the otic vesicle where the position of Cep70 can be seen more clearly; an inset shows one basal body and cilium. Yellow colour, the overlap of Cep70 and acetylated-tubulin signal is due to the angle at which some of the cilia are seen. Again, a long dashed line indicates the apical surface of the eye in (F). Short dashed lines delineate the otic vesicle in (B, C, D and G). (I) and (J) show Cep70 and cilia in the pronephros and spine canal. Again the Cep70 can be seen at the base of cilia. Orientation of the specimens with regard to the embryo is shown by the compass in the panel: A = anterior, P = posterior, D = dorsal, V = ventral. Scale bar: 20 μm.
Mentions: In 24 h.p.f. embryos, GFP-Cep70 localises to the apical surface of cells in the otic vesicle (Fig. 3A) and eye (Fig. 3E) with a punctate appearance. This can be replicated with wholemount immunofluorescence staining of 24 h.p.f. embryos with rabbit anti-Cep70 485-4 (Fig. 3F, G). This staining coincides with that of mouse monoclonal GTU-88 to gamma tubulin which labels centrosomes and basal bodies (Fig. 3B – merge of C and D) and lies at the base of cilia labelled with mouse monoclonal 6-11B-1 to acetylated tubulin (Fig. 3G), shown in detail in Fig. 3H and inset. In the otic vesicle there is non-specific staining of the otoliths but in other ciliated tissues, only the basal bodies are labelled (Fig. 3F (eye), I (pronephros), J (spine canal)). In ciliated cells, therefore, Cep70 localises to the basal bodies of cilia.

Bottom Line: Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase) interacting domain.Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened.We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK. Christopher.Wilkinson@rhul.ac.uk

ABSTRACT

Background: The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms.

Results: We describe zebrafish (Danio rerio) embryos depleted for two uncharacterised, centrosome proteins, Cep70 and Cep131. The phenotype of these embryos resembles that of zebrafish mutants for intraflagellar transport proteins (IFTs), with kidney and ear development affected and left-right asymmetry randomised. These organs and processes are those affected in Bardet-Biedl syndrome and other similar diseases. Like these diseases, the root cause of the phenotype lies, in fact, in dysfunctional cilia, which are shortened but not eliminated in several tissues in the morphants. Centrosomes and basal bodies, on the other hand, are present. Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase) interacting domain. However, we could not detect in yeast two-hybrid assays any interaction with the deacetylase that controls cilium length, HDAC6, or any of the IFTs that we tested.

Conclusion: Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened. We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

Show MeSH
Related in: MedlinePlus