Ift25 is not a cystic kidney disease gene but is required for early steps of kidney development.
ABSTRACT: Eukaryotic cilia are assembled by intraflagellar transport (IFT) where large protein complexes called IFT particles move ciliary components from the cell body to the cilium. Defects in most IFT particle proteins disrupt ciliary assembly and cause mid gestational lethality in the mouse. IFT25 and IFT27 are unusual components of IFT-B in that they are not required for ciliary assembly and mutant mice survive to term. The mutants die shortly after birth with numerous organ defects including duplex kidneys. Completely duplex kidneys result from defects in ureteric bud formation at the earliest steps of metanephric kidney development. Ureteric bud initiation is a highly regulated process involving reciprocal signaling between the ureteric epithelium and the overlying metanephric mesenchyme with regulation by the peri-Wolffian duct stroma. The finding of duplex kidney in Ift25 and Ift27 mutants suggests functions for these genes in regulation of ureteric bud initiation. Typically the deletion of IFT genes in the kidney causes rapid cyst growth in the early postnatal period. In contrast, the loss of Ift25 results in smaller kidneys, which show only mild tubule dilations that become apparent in adulthood. The smaller kidneys appear to result from reduced branching in the developing metanephric kidney. This work indicates that IFT25 and IFT27 are important players in the early development of the kidney and suggest that duplex kidney is part of the ciliopathy spectrum.
Project description:BACKGROUND: Intraflagellar transport (IFT) is the bidirectional movement of IFT particles between the cell body and the distal tip of a flagellum. Organized into complexes A and B, IFT particles are composed of at least 18 proteins. The function of IFT proteins in flagellar assembly has been extensively investigated. However, much less is known about the molecular mechanism of how IFT is regulated. METHODOLOGY/PRINCIPAL FINDINGS: We herein report the identification of a novel IFT particle protein, IFT25, in Chlamydomonas. Dephosphorylation assay revealed that IFT25 is a phosphoprotein. Biochemical analysis of temperature sensitive IFT mutants indicated that IFT25 is an IFT complex B subunit. In vitro binding assay confirmed that IFT25 binds to IFT27, a Rab-like small GTPase component of the IFT complex B. Immunofluorescence staining showed that IFT25 has a punctuate flagellar distribution as expected for an IFT protein, but displays a unique distribution pattern at the flagellar base. IFT25 co-localizes with IFT27 at the distal-most portion of basal bodies, probably the transition zones, and concentrates in the basal body region by partially overlapping with other IFT complex B subunits, such as IFT46. Sucrose density gradient centrifugation analysis demonstrated that, in flagella, the majority of IFT27 and IFT25 including both phosphorylated and non-phosphorylated forms are cosedimented with other complex B subunits in the 16S fractions. In contrast, in cell body, only a fraction of IFT25 and IFT27 is integrated into the preassembled complex B, and IFT25 detected in complex B is preferentially phosphorylated. CONCLUSION/SIGNIFICANCE: IFT25 is a phosphoprotein component of IFT particle complex B. IFT25 directly interacts with IFT27, and these two proteins likely form a subcomplex in vivo. We postulate that the association and disassociation between the subcomplex of IFT25 and IFT27 and complex B might be involved in the regulation of IFT.
Project description:Hair follicle morphogenesis requires precisely controlled reciprocal communications, including hedgehog (Hh) signaling. Activation of the Hh signaling pathway relies on the primary cilium. Disrupting ciliogenesis results in hair follicle morphogenesis defects due to attenuated Hh signaling; however, the loss of cilia makes it impossible to determine whether hair follicle phenotypes in these cilia mutants are caused by the loss of cilia, disruption of Hh signaling, or a combination of these events. In this study, we characterized the function of Ift27, which encodes a subunit of intraflagellar transport (IFT) complex B. Hair follicle morphogenesis of Ift27-null mice was severely impaired, reminiscent of phenotypes observed in cilia and Hh mutants. Furthermore, the Hh signaling pathway was attenuated in Ift27 mutants, which was in association with abnormal ciliary trafficking of SMO and GLI2, and impaired processing of Gli transcription factors; however, formation of the ciliary axoneme was unaffected. The ciliary localization of IFT25 (HSPB11), the binding partner of IFT27, was disrupted in Ift27 mutant cells, and Ift25-null mice displayed hair follicle phenotypes similar to those of Ift27 mutants. These data suggest that Ift27 and Ift25 operate in a genetically and functionally dependent manner during hair follicle morphogenesis. This study suggests that the molecular trafficking machineries underlying ciliogenesis and Hh signaling can be segregated, thereby providing important insights into new avenues of inhibiting Hh signaling, which might be adopted in the development of targeted therapies for Hh-dependent cancers, such as basal cell carcinoma.
Project description:Vertebrate hedgehog signaling is coordinated by the differential localization of the receptors patched-1 and Smoothened in the primary cilium. Cilia assembly is mediated by intraflagellar transport (IFT), and cilia defects disrupt hedgehog signaling, causing many structural birth defects. We generated Ift25 and Ift27 knockout mice and show that they have structural birth defects indicative of hedgehog signaling dysfunction. Surprisingly, ciliary assembly is not affected, but abnormal hedgehog signaling is observed in conjunction with ciliary accumulation of patched-1 and Smoothened. Similarly, Smoothened accumulates in cilia on cells mutated for BBSome components or the BBS binding protein/regulator Lztfl1. Interestingly, the BBSome and Lztfl1 accumulate to high levels in Ift27 mutant cilia. Because Lztfl1 mutant cells accumulate BBSome but not IFT27, it is likely that Lztfl1 functions downstream of IFT27 to couple the BBSome to the IFT particle for coordinated removal of patched-1 and Smoothened from cilia during hedgehog signaling.
Project description:Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2? axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27.
Project description:Intraflagellar transport (IFT) particles are composed of polyprotein complexes IFT-A and IFT-B as well as cargo adaptors such as the BBSome. Two IFT-B subunits, IFT25 and IFT27 were found to form a heterodimer, which is essential in exporting the BBSome out of the cilium but not involved in flagellar assembly and cytokinesis in vertebrates. Controversial results were, however, recorded to show that defects in IFT, flagellar assembly and even cytokinesis were caused by IFT27 knockdown in Chlamydomonas reinhardtii Using C. reinhardtii as a model organism, we report that depletion of IFT25 has no effect on flagellar assembly and does not affect the entry of the BBSome into the flagellum, but IFT25 depletion did impair BBSome movement out of the flagellum, clarifying the evolutionally conserved role of IFT25 in regulating the exit of the BBSome from the flagellum cross species. Interestingly, depletion of IFT25 causes dramatic reduction of IFT27 as expected, which does not cause defects in flagellar assembly and cytokinesis in C. reinhardtii Our data thus support that Chlamydomonas IFT27, like its vertebrate homologues, is not involved in flagellar assembly and cytokinesis.
Project description:Mutations in the human NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) are associated with a rare form of the juvenile renal cystic disease, nephronophthisis type 9, and mutations in murine Nek8 cause renal cysts in jck mice. Cystogenesis involves dysfunctional ciliary signaling, and we have previously reported that Nek8 localizes to the primary cilium in mouse kidney epithelial cells. We now report that in developing mouse kidney, Nek8 is detected in the cilia of a subset of ureteric-bud-derived tubules at embryonic day (E)15.5. An increasing proportion of ureteric-bud-derived tubules express ciliary Nek8 until E18.5. Postnatal day 1 and 7 Nek8 is observed with equal frequency in both ureteric-bud and non-ureteric-bud-derived tubules. To investigate the cell biological consequences of kinase-deficient and jck mutant forms of Nek8, we transiently expressed green fluorescent protein (GFP)-tagged constructs in vitro. Mutations in the kinase and C-terminal domains of Nek8 adversely affected ciliary targeting but did not affect ciliogenesis or ciliary length. Consistent with these in vitro observations, kidneys from homozygous jck mice revealed reduced ciliary expression of Nek8 compared with kidneys from heterozygous (unaffected) mice. These data indicate that the ciliary localization of Nek8 in a subset of ureteric-bud-derived kidney tubules is essential for maintaining the integrity of those tubules in the mammalian kidney.
Project description:The planar cell polarity (PCP) pathway, incorporating non-canonical Wnt signalling, controls embryonic convergent (CE) extension, polarized cell division and ciliary orientation. It also limits diameters of differentiating renal tubules, with mutation of certain components of the pathway causing cystic kidneys. Mutations in mouse Vangl genes encoding core PCP proteins cause neural tube defects (NTDs) and Vangl2 mutations also impair branching of embryonic mouse lung airways. Embryonic metanephric kidneys also undergo branching morphogenesis and Vangl2 is known to be expressed in ureteric bud/collecting duct and metanephric mesenchymal/nephron lineages. These observations led us to investigate metanephroi in Vangl2 mutant mice, Loop-tail (Lp). Although ureteric bud formation is normal in Vangl2(Lp/Lp) embryos, subsequent in vivo and in vitro branching morphogenesis is impaired. Null mutant kidneys are short, consistent with a CE defect. Differentiating glomerular epithelia express several PCP genes (Vangl1/2, Celsr1, Scrib, Mpk1/2 and Fat4) and glomeruli in Vangl2(Lp/Lp) fetuses are smaller and contain less prominent capillary loops than wild-type littermates. Furthermore, Vangl2(Lp/+) kidneys had modest reduction in glomerular numbers postnatally. Vangl2(Lp/Lp) metanephroi contained occasional dilated tubules but no overt cystic phenotype. These data show for the first time that a PCP gene is required for normal morphogenesis of both the ureteric bud and metanephric mesenchyme-derived structures. It has long been recognized that certain individuals with NTDs are born with malformed kidneys, and recent studies have discovered VANGL mutations in some NTD patients. On the basis of our mutant mouse study, we suggest that PCP pathway mutations should be sought when NTD and renal malformation co-exist.
Project description:The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.
Project description:The Wnt5a null mouse is a complex developmental model which, among its several posterior-localized axis defects, exhibits multiple kidney phenotypes, including duplex kidney and loss of the medullary zone. We previously reported that ablation of Wnt5a in nascent mesoderm causes duplex kidney formation as a result of aberrant development of the nephric duct and abnormal extension of intermediate mesoderm. However, these mice also display a loss of the medullary region late in gestation. We have now genetically isolated duplex kidney formation from the medullary defect by specifically targeting the progenitors for both the ureteric bud and metanephric mesenchyme. The conditional mutants fail to form a normal renal medulla but no longer exhibit duplex kidney formation. Approximately 1/3 of the mutants develop hydronephrosis in the kidneys either uni- or bilaterally when using Dll1Cre. The abnormal kidney phenotype becomes prominent at E16.5, which approximates the time when urine production begins in the mouse embryonic kidney, and is associated with a dramatic increase in apoptosis only in mutant kidneys with hydronephrosis. Methylene blue dye injection and histologic examination reveal that aberrant cell death likely results from urine toxicity due to an abnormal ureter-bladder connection. This study shows that Wnt5a is not required for development of the renal medulla and that loss of the renal medullary region in the Wnt5a-deleted kidney is caused by an abnormal ureter-bladder connection.
Project description:Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.