The stumpy gene is required for mammalian ciliogenesis.
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ABSTRACT:
SUBMITTER: Town T
PROVIDER: S-EPMC2268549 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:UNLABELLED: BACKGROUND:Clusterin associated protein 1 (CLUAP1) was initially characterized as a protein that interacts with clusterin, and whose gene is frequently upregulated in colon cancer. Although the consequences of these observations remain unclear, research of CLUAP1 homologs in C. elegans and zebrafish indicates that it is needed for cilia assembly and maintenance in these models. To begin evaluating whether Cluap1 has an evolutionarily conserved role in cilia in mammalian systems and to explore the association of Cluap1 with disease pathogenesis and developmental abnormalities, we generated Cluap1 mutant mice. METHODS:Cluap1 mutant embryos were generated and examined for gross morphological and anatomical defects using light microscopy. Reverse transcription PCR, ?-galactosidase staining assays, and immunofluorescence analysis were used to determine the expression of the gene and localization of the protein in vivo and in cultured cell lines. We also used immunofluorescence analysis and qRT-PCR to examine defects in the Sonic hedgehog signaling pathway in mutant embryos. RESULTS:Cluap1 mutant embryos die in mid-gestation, indicating that it is necessary for proper development. Mutant phenotypes include a failure of embryonic turning, an enlarged pericardial sac, and defects in neural tube development. Consistent with the diverse phenotypes, Cluap1 is widely expressed. Furthermore, the Cluap1 protein localizes to primary cilia, and mutant embryos were found to lack cilia at embryonic day 9.5. The phenotypes observed in Cluap1 mutant mice are indicative of defects in Sonic hedgehog signaling. This was confirmed by analyzing hedgehog signaling activity in Cluap1 mutants, which revealed that the pathway is repressed. CONCLUSIONS:These data indicate that the function of Cluap1 is evolutionarily conserved with regard to ciliogenesis. Further, the results implicate mammalian Cluap1 as a key regulator of hedgehog signaling and as an intraflagellar transport B complex protein. Future studies on mammalian Cluap1 utilizing this mouse model may provide insights into the role for Cluap1 in intraflagellar transport and the association with colon cancer and cystic kidney disorders.
| S-EPMC3556011 | biostudies-literature
Project description:Defects in cilia cause a spectrum of diseases known as ciliopathies. Nephronophthisis, a ciliopathy, is the most common genetic cause of renal disease. Here, I cloned and analysed a nephronophthisis-related gene ift-139 in Caenorhabditis elegans. ift-139 was exclusively expressed in ciliated neurons in C. elegans. Genetic and cellular analyses suggest that ift-139 plays a role in retrograde intraflagellar transport and is required for cilia formation. A homologous point mutation that causes ciliopathy disrupted the function of ift-139 in C. elegans. ift-139 is an orthologue of human TTC21B, mutations in which are known to cause nephronophthisis 12 and short-rib thoracic dysplasia 4. These results suggest that ift-139 is evolutionarily conserved and fundamental to the formation of cilia.
| S-EPMC4981862 | biostudies-literature
Project description:The mammalian retromer consists of subunits VPS26 (either VPS26A or VPS26B), VPS29 and VPS35, and a loosely associated sorting nexin (SNX) heterodimer or a variety of other SNX proteins. Despite involvement in yeast and mammalian cell trafficking, the role of retromer in development is poorly understood, and its impact on primary ciliogenesis remains unknown. Using CRISPR/Cas9 editing, we demonstrate that vps-26-knockout worms have reduced brood sizes, impaired vulval development and decreased body length, all of which have been linked to ciliogenesis defects. Although preliminary studies did not identify worm ciliary defects, and impaired development limited additional ciliogenesis studies, we turned to mammalian cells to investigate the role of retromer in ciliogenesis. VPS35 localized to the primary cilium of mammalian cells, and depletion of VPS26, VPS35, VPS29, SNX1, SNX2, SNX5 or SNX27 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the centriolar protein, CP110 (also known as CCP110), and was required for its removal from the mother centriole. Herein, we characterize new roles for retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, suggesting a novel role for retromer in CP110 removal from the mother centriole.
| S-EPMC9189432 | biostudies-literature
Project description:In Caenorhabditis elegans, the RFX (Daf19) transcription factor is a major regulator of ciliogenesis, controlling the expression of the many essential genes required for making cilia. In vertebrates, however, seven RFX genes have been identified. Bioinformatic analysis suggests that Rfx2 is among the closest homologues of Daf19. We therefore hypothesize that Rfx2 broadly controls ciliogenesis during vertebrate development. Indeed, here we show that Rfx2 in Xenopus is expressed preferentially in ciliated tissues, including neural tube, gastrocoel roof plate, epidermal multi-ciliated cells, otic vesicles, and kidneys. Knockdown of Rfx2 results in cilia-defective embryonic phenotypes and fewer or truncated cilia are observed in Rfx2 morphants. These results indicate that Rfx2 is broadly required for ciliogenesis in vertebrates. Furthermore, we show that Rfx2 is essential for expression of several ciliogenic genes, including TTC25, which we show here is required for ciliogenesis, HH signaling, and left-right patterning.
| S-EPMC3640985 | biostudies-literature
Project description:The pathways controlling cilium biogenesis in different cell types have not been fully elucidated. We recently identified peptidylglycine ?-amidating monooxygenase (PAM), an enzyme required for generating amidated bioactive signaling peptides, in Chlamydomonas and mammalian cilia. Here, we show that PAM is required for the normal assembly of motile and primary cilia in Chlamydomonas, planaria and mice. Chlamydomonas PAM knockdown lines failed to assemble cilia beyond the transition zone, had abnormal Golgi architecture and altered levels of cilia assembly components. Decreased PAM gene expression reduced motile ciliary density on the ventral surface of planaria and resulted in the appearance of cytosolic axonemes lacking a ciliary membrane. The architecture of primary cilia on neuroepithelial cells in Pam-/- mouse embryos was also aberrant. Our data suggest that PAM activity and alterations in post-Golgi trafficking contribute to the observed ciliogenesis defects and provide an unanticipated, highly conserved link between PAM, amidation and ciliary assembly.
| S-EPMC5461114 | biostudies-literature
Project description:Serum starvation stimulates cilia growth in cultured cells, yet serum factors associated with ciliogenesis are unknown. Previously, we showed that starvation induces rapid Rab11-dependent vesicular trafficking of Rabin8, a Rab8 guanine-nucleotide exchange factor (GEF), to the mother centriole, leading to Rab8 activation and cilium growth. Here, we demonstrate that through the LPA receptor 1 (LPAR1), serum lysophosphatidic acid (LPA) inhibits Rab11a-Rabin8 interaction and ciliogenesis. LPA/LPAR1 regulates ciliogenesis initiation via downstream PI3K/Akt activation, independent of effects on cell cycle. Akt stabilizes Rab11a binding to its effector, WDR44, and a WDR44-pAkt-phosphomimetic mutant blocks ciliogenesis. WDR44 depletion promotes Rabin8 preciliary trafficking and ciliogenesis-initiating events at the mother centriole. Our work suggests disruption of Akt signaling causes a switch from Rab11-WDR44 to the ciliogenic Rab11-FIP3-Rabin8 complex. Finally, we demonstrate that Akt regulates downstream ciliogenesis processes associated with Rab8-dependent cilia growth. Together, this study uncovers a mechanism whereby serum mitogen signaling regulates Rabin8 preciliary trafficking and ciliogenesis initiation.
| S-EPMC7457226 | biostudies-literature
Project description:Mutations affecting ciliary components cause ciliopathies. As described here, we investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel and Joubert syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2 and Cc2d2a. Components of this complex co-localize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes Joubert syndrome. Thus, a transition zone complex of Meckel and Joubert syndrome proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.
| S-EPMC3145011 | biostudies-literature
Project description:We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5, a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins.
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Project description:More than blood (Mtb) is a novel gene that is widely expressed in mouse embryos prior to gastrulation but is subsequently restricted to specific tissues, including the developing central nervous system and hematopoietic organs. Since MTB is highly expressed in the fetal liver and developing thymus, we predicted that MTB would be required for hematopoiesis and that embryos deficient in MTB would die of anemia. Surprisingly, embryos with a targeted disruption of Mtb died prior to the initiation of blood cell development, immediately following implantation. This lethality is due to a defect in expansion of the inner cell mass (ICM), as Mtb(-/-) blastocysts failed to exhibit outgrowth of the ICM, both in vitro and in vivo. Furthermore, Mtb(-/-) blastocysts exhibited a higher frequency of apoptotic cells than wild-type or heterozygous blastocysts. These findings demonstrate that Mtb is a novel gene that is essential for early embryonic development.
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Project description: Not available
| S-EPMC8419681 | biostudies-literature