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Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling.

Yee LE, Garcia-Gonzalo FR, Bowie RV, Li C, Kennedy JK, Ashrafi K, Blacque OE, Leroux MR, Reiter JF - PLoS Genet. (2015)

Bottom Line: Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did.However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1.Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may participate in their pathogenesis. The ciliary transition zone contains two protein complexes affected in the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP). The BBSome is a third protein complex, affected in the ciliopathy Bardet-Biedl syndrome (BBS). We tested whether mutations in MKS, NPHP and BBS complex genes modify the phenotypic consequences of one another in both C. elegans and mice. To this end, we identified TCTN-1, the C. elegans ortholog of vertebrate MKS complex components called Tectonics, as an evolutionarily conserved transition zone protein. Neither disruption of TCTN-1 alone or together with MKS complex components abrogated ciliary structure in C. elegans. In contrast, disruption of TCTN-1 together with either of two NPHP complex components, NPHP-1 or NPHP-4, compromised ciliary structure. Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did. As in nematodes, disrupting two components of the mouse MKS complex did not cause additive phenotypes compared to single mutants. However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1. Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.

No MeSH data available.


Related in: MedlinePlus

Mouse Tctn1 and Nphp4 have distinct roles in transition zone composition and overlapping roles in ciliogenesis.(A) Limb bud sections (left) from E11.5 embryos were stained for acetylated tubulin (TubAc, green) to mark the ciliary axonemes, γ-tubulin (red) to mark the basal bodies and DAPI (blue) to mark nuclei. Scale bar, 5 μm. Transmission electron microscopy (TEM, right) for each genotype. Scale bar, 200 nm. (B) Quantification of limb bud cilia in control, Tctn1-/-Nphp4n/+, and Tctn1-/-Nphp4n/n mutants from TEM fields of view. Error bars represent the standard error of the mean. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.001). (C) Fibroblasts derived from E13.5 limb buds of the indicated genotypes stained for TubAc (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). White arrowheads indicate cilia in Tctn1-/-Nphp4n/+ and Tctn1-/-Nphp4n/n mutants. Scale bar, 10 μm. (D) Quantification of proportion of ciliated cultured limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.0001). (E) Immunostaining of limb bud fibroblasts for Nphp1 (red), TubAc (green), and γ-tubulin (cyan). Scale bar, 1 μm.
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pgen.1005627.g006: Mouse Tctn1 and Nphp4 have distinct roles in transition zone composition and overlapping roles in ciliogenesis.(A) Limb bud sections (left) from E11.5 embryos were stained for acetylated tubulin (TubAc, green) to mark the ciliary axonemes, γ-tubulin (red) to mark the basal bodies and DAPI (blue) to mark nuclei. Scale bar, 5 μm. Transmission electron microscopy (TEM, right) for each genotype. Scale bar, 200 nm. (B) Quantification of limb bud cilia in control, Tctn1-/-Nphp4n/+, and Tctn1-/-Nphp4n/n mutants from TEM fields of view. Error bars represent the standard error of the mean. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.001). (C) Fibroblasts derived from E13.5 limb buds of the indicated genotypes stained for TubAc (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). White arrowheads indicate cilia in Tctn1-/-Nphp4n/+ and Tctn1-/-Nphp4n/n mutants. Scale bar, 10 μm. (D) Quantification of proportion of ciliated cultured limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.0001). (E) Immunostaining of limb bud fibroblasts for Nphp1 (red), TubAc (green), and γ-tubulin (cyan). Scale bar, 1 μm.

Mentions: The genetic interaction of Tctn1 and Nphp4 suggested that the mammalian MKS and NPHP complexes have partially overlapping ciliary functions. To investigate how these two complexes participate in ciliary functions, we examined cilia in the forelimb, a tissue phenotypically affected in Tctn1-/-Nphp4n/n double mutants. Immunostaining revealed that ciliation in Tctn1-/- forelimbs was reduced compared to controls (Fig 6A). Tctn1-/-Nphp4n/n forelimbs were further depleted of cilia compared to Tctn1-/- forelimbs (Fig 6A). Transmission electron microscopy confirmed the depletion of cilia in the forelimb buds of Tctn1-/-Nphp4n/n double mutants, which possessed docked basal bodies, but lacked ciliary axonemes (Fig 6A and 6B). The greater ciliogenesis defect in Tctn1-/-Nphp4n/n forelimbs likely accounts for the increased polydactyly, as cilia are required for Shh-dependent patterning of the limb buds [57–59].


Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling.

Yee LE, Garcia-Gonzalo FR, Bowie RV, Li C, Kennedy JK, Ashrafi K, Blacque OE, Leroux MR, Reiter JF - PLoS Genet. (2015)

Mouse Tctn1 and Nphp4 have distinct roles in transition zone composition and overlapping roles in ciliogenesis.(A) Limb bud sections (left) from E11.5 embryos were stained for acetylated tubulin (TubAc, green) to mark the ciliary axonemes, γ-tubulin (red) to mark the basal bodies and DAPI (blue) to mark nuclei. Scale bar, 5 μm. Transmission electron microscopy (TEM, right) for each genotype. Scale bar, 200 nm. (B) Quantification of limb bud cilia in control, Tctn1-/-Nphp4n/+, and Tctn1-/-Nphp4n/n mutants from TEM fields of view. Error bars represent the standard error of the mean. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.001). (C) Fibroblasts derived from E13.5 limb buds of the indicated genotypes stained for TubAc (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). White arrowheads indicate cilia in Tctn1-/-Nphp4n/+ and Tctn1-/-Nphp4n/n mutants. Scale bar, 10 μm. (D) Quantification of proportion of ciliated cultured limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.0001). (E) Immunostaining of limb bud fibroblasts for Nphp1 (red), TubAc (green), and γ-tubulin (cyan). Scale bar, 1 μm.
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pgen.1005627.g006: Mouse Tctn1 and Nphp4 have distinct roles in transition zone composition and overlapping roles in ciliogenesis.(A) Limb bud sections (left) from E11.5 embryos were stained for acetylated tubulin (TubAc, green) to mark the ciliary axonemes, γ-tubulin (red) to mark the basal bodies and DAPI (blue) to mark nuclei. Scale bar, 5 μm. Transmission electron microscopy (TEM, right) for each genotype. Scale bar, 200 nm. (B) Quantification of limb bud cilia in control, Tctn1-/-Nphp4n/+, and Tctn1-/-Nphp4n/n mutants from TEM fields of view. Error bars represent the standard error of the mean. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.001). (C) Fibroblasts derived from E13.5 limb buds of the indicated genotypes stained for TubAc (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). White arrowheads indicate cilia in Tctn1-/-Nphp4n/+ and Tctn1-/-Nphp4n/n mutants. Scale bar, 10 μm. (D) Quantification of proportion of ciliated cultured limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t-tests (* p<0.05; ** p<0.0001). (E) Immunostaining of limb bud fibroblasts for Nphp1 (red), TubAc (green), and γ-tubulin (cyan). Scale bar, 1 μm.
Mentions: The genetic interaction of Tctn1 and Nphp4 suggested that the mammalian MKS and NPHP complexes have partially overlapping ciliary functions. To investigate how these two complexes participate in ciliary functions, we examined cilia in the forelimb, a tissue phenotypically affected in Tctn1-/-Nphp4n/n double mutants. Immunostaining revealed that ciliation in Tctn1-/- forelimbs was reduced compared to controls (Fig 6A). Tctn1-/-Nphp4n/n forelimbs were further depleted of cilia compared to Tctn1-/- forelimbs (Fig 6A). Transmission electron microscopy confirmed the depletion of cilia in the forelimb buds of Tctn1-/-Nphp4n/n double mutants, which possessed docked basal bodies, but lacked ciliary axonemes (Fig 6A and 6B). The greater ciliogenesis defect in Tctn1-/-Nphp4n/n forelimbs likely accounts for the increased polydactyly, as cilia are required for Shh-dependent patterning of the limb buds [57–59].

Bottom Line: Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did.However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1.Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may participate in their pathogenesis. The ciliary transition zone contains two protein complexes affected in the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP). The BBSome is a third protein complex, affected in the ciliopathy Bardet-Biedl syndrome (BBS). We tested whether mutations in MKS, NPHP and BBS complex genes modify the phenotypic consequences of one another in both C. elegans and mice. To this end, we identified TCTN-1, the C. elegans ortholog of vertebrate MKS complex components called Tectonics, as an evolutionarily conserved transition zone protein. Neither disruption of TCTN-1 alone or together with MKS complex components abrogated ciliary structure in C. elegans. In contrast, disruption of TCTN-1 together with either of two NPHP complex components, NPHP-1 or NPHP-4, compromised ciliary structure. Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did. As in nematodes, disrupting two components of the mouse MKS complex did not cause additive phenotypes compared to single mutants. However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1. Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.

No MeSH data available.


Related in: MedlinePlus