The primary cilium is an antenna-like structure that protrudes from the cell surface of quiescent/differentiated cells and participates in extracellular signal processing [1][2][3] . Here, we report that mice deficient for the lipid 5-phosphatase Inpp5e develop a multiorgan disorder associated with structural defects of the primary cilium. In ciliated mouse embryonic fibroblasts, Inpp5e is concentrated in the axoneme of the primary cilium. Inpp5e inactivation did not impair ciliary assembly but altered the stability of pre-established cilia after serum addition. Blocking phosphoinositide 3-kinase (PI3K) activity or ciliary platelet-derived growth factor receptor a (PDGFRa) restored ciliary stability. In human INPP5E, we identified a mutation affecting INPP5E ciliary localization and cilium stability in a family with MORM syndrome, a condition related to Bardet-Biedl syndrome. Together, our results show that INPP5E plays an essential role in the primary cilium by controlling ciliary growth factor and PI3K signaling and stability, and highlight the consequences of INPP5E dysfunction.Lipid 5-phosphatases selectively remove the phosphate from position D-5 of the inositol ring of phosphoinositides and inositolphosphates 4,5 . To characterize the functions of the 5-phosphatase Inpp5e 6-8 , we generated Inpp5e D/+ mice ( Supplementary Fig. 1a). We obtained no adult Inpp5e D/D mutant mice from intercrosses between Inpp5e D/+ mice. However, at embryonic day 13.5 (E13.5) and E18.5, 16.9% (11/65) and 14.8% (12/81) of embryos were homozygous for the deletion allele, respectively. The mutant mice died soon after birth, indicating that total inactivation of Inpp5e led to embryonic and postnatal death. Analyses confirmed the absence of Inpp5e protein in mutant cells and tissues (Fig. 1a). Inpp5e D/D mice presented with bilateral anophthalmos (100%, n ¼ 43) and postaxial hexadactyly (62.5%, n ¼ 16; Fig. 1b,c). Histological analyses revealed that eye development ceased at the optic vesicle stage, just before the appearance of the optic cup (Fig. 1d). Analysis of kidneys from the mice revealed the presence of multiple cysts (100%, n ¼ 10; Fig. 1e). Of the cysts, 84% expressed AQP2 and 14% expressed AQP1, indicating an origin in cortical collecting and connecting ducts (when AQP2 + ) as well as proximal tubules and the descending limb of the loop of Henle (when AQP1 + ) ( Supplementary Fig. 2). Only 2% of the renal glomeruli were cystic. Inpp5e D/D embryos had skeletal abnormalities such as a bifid sternum (50%, n ¼ 6), delayed ossification of metacarpals and phalanges (100%, n ¼ 5) and cleft palate (75%, n ¼ 4; Fig. 1f-h). We identified cerebral developmental defects, such as anencephaly and exencephaly, in 30% of Inpp5e D/D embryos at E15.5 (n ¼ 30; Fig. 1i,j). We did not detect liver alterations, laterality defects or respiratory cilium defects in mutant animals. The tissue localization of lesions observed in Inpp5e D/D embryos matched the tissue expression of Inpp5e mRNA during mouse embryogenesis ( Supplementary Fig. 3).Becau...

Phosphotidylinositol (PtdIns) signaling is tightly regulated, both spatially and temporally, by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events 1. Joubert Syndrome (JS) characterized by a specific midbrain-hindbrain malformation (“molar tooth sign”) and variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly 2, and is included in the newly emerging group of “ciliopathies”. In patients linking to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected in JS, and mutations promoted premature destabilization of cilia in response to stimulation. Thus, these data links PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly appreciated for its role in mediating cell signals and neuronal function.

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DNA methylation regulates gene expression in a cell-type specific way. Although peripheral blood mononuclear cells (PBMCs) comprise a heterogeneous cell population, most studies of DNA methylation in blood are performed on total mononuclear cells. In this study, we investigated high resolution methylation profiles of 58 CpG sites dispersed over eight immune response genes in multiple purified blood cells from healthy adults and newborns. Adjacent CpG sites showed methylation levels that were increasingly correlated in adult blood vs. cord blood. Thus, while interindividual variability increases from newborn to adult blood, the underlying methylation changes may not be merely stochastic, but seem to be orchestrated as clusters of adjacent CpG sites. Multiple linear regression analysis showed that interindividual methylation variability was influenced by distance of average methylation levels to the closest border (0 or 100%), presence of transcription factor binding sites, CpG conservation across species and age. Furthermore, CD4+ and CD14+ cell types were negative predictors of methylation variability. Concerns that PBMC methylation differences may be confounded by variations in blood cell composition were justified for CpG sites with large methylation differences across cell types, such as in the IFN-γ gene promoter. Taken together, our data suggest that unsorted mononuclear cells are reasonable surrogates of CD8+ and, to a lesser extent, CD4+ T cell methylation in adult peripheral, but not in neonatal, cord blood.

Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3(-/-) corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3(-/-) VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development.