Intraflagellar transport (IFT) proteins were first identified as essential factors for the growth and maintenance of flagella in the single-celled alga Chlamydomonas reinhardtii. In a screen for embryonic patterning mutations induced by ethylnitrosourea, here we identify two mouse mutants, wimple (wim) and flexo (fxo), that lack ventral neural cell types and show other phenotypes characteristic of defects in Sonic hedgehog signalling. Both mutations disrupt IFT proteins: the wim mutation is an allele of the previously uncharacterized mouse homologue of IFT172; and fxo is a new hypomorphic allele of polaris, the mouse homologue of IFT88. Genetic analysis shows that Wim, Polaris and the IFT motor protein Kif3a are required for Hedgehog signalling at a step downstream of Patched1 (the Hedgehog receptor) and upstream of direct targets of Hedgehog signalling. Our data show that IFT machinery has an essential and vertebrate-specific role in Hedgehog signal transduction.

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently leads to renal failure. Mutations in polycystin-1 (PC1) underlie most cases of ADPKD, but the function of PC1 has remained poorly understood. No preventive treatment for this disease is available. Here, we show that the cytoplasmic tail of PC1 interacts with tuberin, and the mTOR pathway is inappropriately activated in cyst-lining epithelial cells in human ADPKD patients and mouse models. Rapamycin, an inhibitor of mTOR, is highly effective in reducing renal cystogenesis in two independent mouse models of PKD. Treatment of human ADPKD transplant-recipient patients with rapamycin results in a significant reduction in native polycystic kidney size. These results indicate that PC1 has an important function in the regulation of the mTOR pathway and that this pathway provides a target for medical therapy of ADPKD.rapamycin ͉ renal epithelial cells ͉ tuberin A utosomal-dominant polycystic kidney disease (ADPKD) is one of the most common human monogenic diseases and affects 12 million people worldwide (for recent reviews, see refs.

In the adult brain, neuroblasts born in the subventricular zone migrate from the walls of the lateral ventricles to the olfactory bulb. How do these cells orient over such a long distance and through complex territories? Here we show that neuroblast migration parallels cerebrospinal fluid (CSF) flow. Beating of ependymal cilia is required for normal CSF flow, concentration gradient formation of CSF guidance molecules, and directional migration of neuroblasts. Results suggest that polarized epithelial cells contribute important vectorial information for guidance of young, migrating neurons.

Polycystic kidney disease (PKD) includes a group of disorders that are characterized by the presence of cysts in the kidney and other organs,including the pancreas. Here we show that in orpk mice, a model system for PKD that harbors a mutation in the gene that encodes the polaris protein, pancreatic defects start to occur at the end of gestation, with an initial expansion of the developing pancreatic ducts. Ductal dilation continues rapidly after birth and results in the formation of large,interconnected cysts. Expansion of pancreatic ducts is accompanied by apoptosis of neighboring acinar cells, whereas endocrine cell differentiation and islet formation appears to be unaffected. Polaris has been shown to co-localize with primary cilia, and these structures have been implicated in the formation of renal cysts. In the orpk pancreas, cilia numbers are reduced and cilia length is decreased. Expression of polycystin-2, a protein involved in PKD, is mislocalized in orpk mice. Furthermore, the cellular localization of β-catenin, a protein involved in cell adhesion and Wnt signaling, is altered. Thus, polaris and primary cilia function are required for the maturation and maintenance of proper tissue organization in the pancreas.

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the progressive dilatation of collecting ducts, the nephron segments responsible for the final renal regulation of sodium, potassium, acid-base, and water balance. Murine models of ARPKD possess mutations in genes encoding cilia-associated proteins, including Tg737 in orpk mice. New findings implicate defects in structure/function of primary cilia as central to the development of polycystic kidney disease. Our group (Liu W, Xu S, Woda C, Kim P, Weinbaum S, and Satlin LM, Am J Physiol Renal Physiol 285: F998-F1012, 2003) recently reported that increases in luminal flow rate in rabbit collecting ducts increase intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells therein. We thus hypothesized that fluid shear acting on the apical membrane or hydrodynamic bending moments acting on the cilium increase renal epithelial [Ca(2+)](i). To further explore this, we tested whether flow-induced [Ca(2+)](i) transients in collecting ducts from mutant orpk mice, which possess structurally abnormal cilia, differ from those in controls. Isolated segments from 1- and 2-wk-old mice were microperfused in vitro and loaded with fura 2; [Ca(2+)](i) was measured by digital ratio fluorometry before and after the rate of luminal flow was increased. All collecting ducts responded to an increase in flow with an increase in [Ca(2+)](i), a response that appeared to be dependent on luminal Ca(2+) entry. However, the magnitude of the increase in [Ca(2+)](i) in 2- but not 1-wk-old mutant orpk animals was blunted. We speculate that this defect in mechano-induced Ca(2+) signaling in orpk mice leads to aberrant structure and function of the collecting duct in ARPKD.

Tg737 mutant mice exhibit pathologic conditions in numerous tissues along with skeletal patterning defects. Herein, we characterize the skeletal pathologic conditions and confirm a role for Tg737 in skeletal patterning through transgenic rescue. Analyses were conducted in both the hypomorphic Tg737 orpk allele that results in duplication of digit one and in the null Tg737 ⌬2-3␤Gal allele that is an embryonic lethal mutation exhibiting eight digits per limb. In early limb buds, Tg737 expression is detected throughout the mesenchyme becoming concentrated in precartilage condensations at later stages. In situ analyses indicate that the Tg737 orpk mutant limb defects are not associated with changes in expression of Shh, Ihh, HoxD11-13, Patched, BMPs, or Glis. Likewise, in Tg737 ⌬2-3␤Gal mutant embryos, there was no change in Shh expression. However, in both alleles, Fgf4 was ectopically expressed on the anterior apical ectodermal ridge. Collectively, the data argue for a dosage effect of Tg737 on the limb phenotypes and that the polydactyly is independent of Shh misexpression. Developmental Dynamics 227:78 -90, 2003.

Polycystic kidney diseases are characterized by the progressive expansion of multiple cystic lesions, which compromise the function of normal parenchyma. Throughout the course of these diseases, renal tubular function and structure are altered, changing the tubular microenvironment and ultimately causing the formation and progressive expansion of cystic lesions. Renal tubules are predisposed to cystogenesis when a germ line mutation is inherited in either the human PKD1 or PKD2 genes in autosomal dominant polycystic kidney disease (ADPKD) or when a homozygous mutation in Tg737 is inherited in the orpk mouse model of autosomal recessive polycystic kidney disease (ARPKD). Recent information strongly suggests that the protein products of these disease genes may form a macromolecular signaling structure, the polycystin complex, which regulates fundamental aspects of renal epithelial development and cell biology. Here, we re-examine the cellular pathophysiology of renal cyst formation and enlargement in the context of our current understanding of the molecular genetics of ADPKD and ARPKD.

While this data is consistent with a role for polaris in ciliogenesis, it does not support a direct connection between ciliogenesis and cystic disease. Similarities in cyst formation and striking differences in cilia expression associated with these ARPKD mouse models indicates that cyst formation and cilia expression are independent phenotypic features regulated by polaris.