The planar cell polarity (PCP) signaling pathway is essential for embryonic development because it governs diverse cellular behaviors, and the “core PCP” proteins, such as Dishevelled and Frizzled, have been extensively characterized1–4. By contrast, the “PCP effector” proteins, such as Intu and Fuz, remain largely unstudied5, 6. These proteins are essential for PCP signaling, but they have never been investigated in a mammal and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice display neural tube defects, skeletal dysmorphologies, and Hedgehog signaling defects stemming from disrupted ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we establish a central role for Fuz in membrane trafficking, showing that Fuz is essential for trafficking of cargo to basal bodies and to the apical tips of cilia. Fuz is also essential for exocytosis in secretory cells. Finally, we identify a novel, Rab-related small GTPase as a Fuz interaction partner that is also essential for ciliogenesis and secretion. These results are significant because they provide novel insights into the mechanisms by which developmental regulatory systems like PCP signaling interface with fundamental cellular systems such as the vesicle trafficking machinery.

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SummaryCiliopathies are a broad class of human disorders with craniofacial dysmorphology as a common feature. Among these is high arched palate, a condition that affects speech and quality of life. Using the ciliopathic Fuz mutant mouse, we find that high arched palate does not, as commonly suggested, arise from midface hypoplasia. Rather, increased neural crest expands the maxillary primordia. In Fuz mutants, this phenotype stems from dysregulated Gli processing, which in turn results in excessive craniofacial Fgf8 gene expression. Accordingly, genetic reduction of Fgf8 ameliorates the maxillary phenotypes. Similar phenotypes result from mutation of oral-facial-digital syndrome 1 (Ofd1), suggesting that aberrant transcription of Fgf8 is a common feature of ciliopathies. High arched palate is also a prevalent feature of fibroblast growth factor (FGF) hyperactivation syndromes. Thus, our findings elucidate the etiology for a common craniofacial anomaly and identify links between two classes of human disease: FGF-hyperactivation syndromes and ciliopathies.

3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor A (ERA) interactions. In this study, we used 3MC and 3,3V,4,4V,5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ERA but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ERA, and this was confirmed in a competitive ERA binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ERA/YFP-ERA interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ERA (but not AhR) association with the estrogenresponsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ERA-dependent transactivation and extend the number of ligands that activate both AhR and ERA. (Cancer Res 2006; 66(4): 2459-67)

Many antiepileptic drugs (AEDs) have therapeutic applications that extend beyond epilepsy to include neuropathic pain, migraine headaches and psychiatric disorders. The risk of some AEDs has been clearly established, but for newer drugs, small sample sizes and polytherapy exposures preclude a conclusive determination of their teratogenic potential. Most women with epilepsy will require AED therapy throughout their entire pregnancy to control seizures; the vast majority of pregnancies in women with epilepsy have positive outcomes. A conservative estimate suggests that AED monotherapy doubles, and polytherapy triples, the risk for major congenital malformations. Furthermore, while evidence is still accruing, recent investigations suggest that exposure to select AEDs results in altered cognitive function later in development. There is no evidence to suggest that additional folic acid supplementation ameliorates the increased risk of congenital malformations conferred by in utero AED exposure.

Valproic acid (VPA) is a major antiepileptic drug (AED); however, its use is limited by two life-threatening side effects: teratogenicity and hepatotoxicity. Several constitutional isomers of VPA and their amide and urea derivatives were synthesized and evaluated in three different anticonvulsant animal models and a mouse model for AED-induced teratogenicity. The urea derivatives of three VPA constitutional isomers propylisopropylacetylurea, diisopropylacetylurea, and 2-ethyl-3-methyl-pentanoylurea displayed a broad spectrum of anticonvulsant activity in rats with a clear superiority over their corresponding amides and acids. Enanatiomers of propylisopropylacetylurea and propylisopropylacetamide revealed enantioselective anticonvulsant activity, whereas only enantiomers of propylisopropylacetylurea displayed enantioselective teratogenicity. These potent urea derivatives caused neural tube defects, but only at doses markedly exceeding their effective dose, whereas VPA showed no separation between its anticonvulsant activity and teratogenicity. The broad spectrum of anticonvulsant activity of the urea derivatives coupled with their wide safety margin make them potential candidates to become new, potent AEDs.

sec-Butylpropylacetamide (racemic-SPD) is a chiral CNS-active amide derivative of valproic acid (VPA). This study describes synthesis and stereospecific comparative pharmacodynamics (PD, anticonvulsant activity and teratogenicity) and pharmacokinetic (PK) analysis of four individual SPD stereoisomers. SPD stereoisomers' anticonvulsant activity was comparatively evaluated in several anticonvulsant animal models including the benzodiazepine-resistant status epilepticus (SE). SPD stereoisomers' PK-PD relationship was evaluated in rats. Teratogenicity of SPD stereoisomers was evaluated in SWV mice strain, susceptible to VPA-induced neural tube defect (NTD). SPD stereoisomers (141 or 283 mg/kg) did not cause NTD. SPD has stereoselective PK and PD. (2R,3S)-SPD and (2S,3R)-SPD higher clearance led to a 50% lower plasma exposure that may contribute to their relative lower activity in the pilocarpine-induced SE model. (2S,3S)-SPD, (2R,3R)-SPD, and racemic-SPD have similar anticonvulsant activity and a PK profile that are better than those of (2R,3S)-SPD and (2S,3R)-SPD, making them good candidates for development as new, potent antiepileptics with a potential in benzodiazepine-resistant SE.

Our findings demonstrate for the first time a prominent role of the Folbp1 gene in mediating susceptibility to heart defects.