The skin is a highly regenerative organ which plays critical roles in protecting the body and sen... more The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation
Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function ... more Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3- kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD, however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de ...
Ciliated bronchial epithelium 1 (CBE1) is a microtubule-associated protein localized to the manch... more Ciliated bronchial epithelium 1 (CBE1) is a microtubule-associated protein localized to the manchette and developing flagellum during spermiogenesis, and associated with sperm maturation arrest in humans. It was hypothesized that CBE1 functions in microtubule-mediated transport mechanisms and sperm tail formation. To test this hypothesis, we analysed Cbe1 expression and localization during spermiogenesis, and in mouse IMCD3 cells as a model of ciliogenesis. Further, we generated and analysed the fertility of a Cbe1 mutant mouse line. Mice containing a homozygous deletion in the long forms of Cbe1 were born at a lower frequency than predicted by Mendelian inheritance, however, adult male mice were fertile. An in-depth analysis of the Cbe1 gene revealed alternative transcript variants, which were not affected by the exon 2 mutation. To assess whether short variants compensate for the loss of long variants, exons 2 and 4 (which affect all variants) were individually mutated in IMCD3 ce...
Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joub... more Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joubert and MORM syndromes; however, the role of INPP5E in ciliary biology is not well understood. Here, we describe an interaction between INPP5E and AURKA, a centrosomal kinase that regulates mitosis and ciliary disassembly, and we show that this interaction is important for the stability of primary cilia. Furthermore, AURKA phosphorylates INPP5E and thereby increases its 5-phosphatase activity, which in turn promotes transcriptional downregulation of AURKA, partly through an AKT-dependent mechanism. These findings establish the first direct link between AURKA and phosphoinositide signaling and suggest that the function of INPP5E in cilia is at least partly mediated by its interactions with AURKA.
The skin is a highly regenerative organ which plays critical roles in protecting the body and sen... more The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation
Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function ... more Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3- kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD, however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de ...
Ciliated bronchial epithelium 1 (CBE1) is a microtubule-associated protein localized to the manch... more Ciliated bronchial epithelium 1 (CBE1) is a microtubule-associated protein localized to the manchette and developing flagellum during spermiogenesis, and associated with sperm maturation arrest in humans. It was hypothesized that CBE1 functions in microtubule-mediated transport mechanisms and sperm tail formation. To test this hypothesis, we analysed Cbe1 expression and localization during spermiogenesis, and in mouse IMCD3 cells as a model of ciliogenesis. Further, we generated and analysed the fertility of a Cbe1 mutant mouse line. Mice containing a homozygous deletion in the long forms of Cbe1 were born at a lower frequency than predicted by Mendelian inheritance, however, adult male mice were fertile. An in-depth analysis of the Cbe1 gene revealed alternative transcript variants, which were not affected by the exon 2 mutation. To assess whether short variants compensate for the loss of long variants, exons 2 and 4 (which affect all variants) were individually mutated in IMCD3 ce...
Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joub... more Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joubert and MORM syndromes; however, the role of INPP5E in ciliary biology is not well understood. Here, we describe an interaction between INPP5E and AURKA, a centrosomal kinase that regulates mitosis and ciliary disassembly, and we show that this interaction is important for the stability of primary cilia. Furthermore, AURKA phosphorylates INPP5E and thereby increases its 5-phosphatase activity, which in turn promotes transcriptional downregulation of AURKA, partly through an AKT-dependent mechanism. These findings establish the first direct link between AURKA and phosphoinositide signaling and suggest that the function of INPP5E in cilia is at least partly mediated by its interactions with AURKA.
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Papers by Denny Cottle