Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be nonmotile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates.

Developing organs acquire a specific three-dimensional form that ensures their normal function. Cardiac function, for example, depends upon properly shaped chambers that emerge from a primitive heart tube. The cellular mechanisms that control chamber shape are not yet understood. Here, we demonstrate that chamber morphology develops via changes in cell morphology, and we determine key regulatory influences on this process. Focusing on the development of the ventricular chamber in zebrafish, we show that cardiomyocyte cell shape changes underlie the formation of characteristic chamber curvatures. In particular, cardiomyocyte elongation occurs within a confined area that forms the ventricular outer curvature. Because cardiac contractility and blood flow begin before chambers emerge, cardiac function has the potential to influence chamber curvature formation. Employing zebrafish mutants with functional deficiencies, we find that blood flow and contractility independently regulate cell shape changes in the emerging ventricle. Reduction of circulation limits the extent of cardiomyocyte elongation; in contrast, disruption of sarcomere formation releases limitations on cardiomyocyte dimensions. Thus, the acquisition of normal cardiomyocyte morphology requires a balance between extrinsic and intrinsic physical forces. Together, these data establish regionally confined cell shape change as a cellular mechanism for chamber emergence and as a link in the relationship between form and function during organ morphogenesis.

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Human nicotinic acetylcholine receptor (AChR) subtypes ␣3␤2, ␣3␤2␣5, ␣3␤4, and ␣3␤4␣5 were stably expressed in cells derived from the human embryonic kidney cell line 293. ␣3␤4 AChRs were found in prominent 2-m patches on the cell surface, whereas most ␣3␤2 AChRs were more diffusely distributed. The functional properties of the ␣3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on ␣3␤4 AChRs and nearly a full agonist on ␣3␤2␣5 AChRs. Chronic exposure of cells expressing ␣3␤2 AChRs or ␣3␤2␣5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of ␣3␤4 or ␣3␤4␣5 AChRs, i.e. the up-regulation of ␣3 AChRs depended on the presence of ␤2 but not ␤4 subunits in the AChRs. This was also found to be true of ␣3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, ␣3␤2 AChRs were expressed at much lower levels than ␣3␤4 AChRs, but in the presence of nicotine, the amount of ␣3␤2 AChRs exceeded that of ␣3␤4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated ␣3␤2 AChRs were functional. The nicotinic antagonists curare and dihydro-␤-erythroidine also up-regulated ␣3␤2 AChRs, but only by 3-5-fold. The channel blocker mecamylamine did not cause up-regulation of ␣3␤2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of ␣3␤2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation, and to establish laterality1. Cilia motility defects cause Primary Ciliary Dyskinesia (PCD, MIM 242650), a disorder affecting 1:15-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive cilia bending2. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD linked loci3. Here we show that the zebrafish cilia paralysis mutant schmalhanstn222 (smh) mutant encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a regulated gene. Screening 146 unrelated PCD families identified patients in six families with reduced outer dynein arms, carrying mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103 functions as a tightly bound, axoneme-associated protein. The results identify Ccdc103 as a novel dynein arm attachment factor that when mutated causes Primary Ciliary Dyskinesia.

A mutation (S247F) in the channel-lining domain (M2) of the ␣4 nicotinic acetylcholine receptor (AChR) subunit has previously been linked genetically to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).To better understand the functional significance of this mutation, we characterized the properties of mutant and wild-type human ␣4␤2 AChRs expressed in Xenopus oocytes. Both had similar expression levels and EC 50 values for ACh and nicotine. Substantial use-dependent functional upregulation was found for mutant ␣4␤2 AChRs, but not for wild type. Mutant AChR responses showed faster desensitization, slower recovery from desensitization, less inward rectification, and virtually no Ca 2ϩ permeability as compared with wild-type ␣4␤2 AChRs. Addition of the ␣5 subunit restored Ca 2ϩ permeability to the mutant ␣4␤2␣5 AChRs. At Ϫ80 mV, wild-type ␣4␤2 AChR single channel currents exhibited two conductances, each with two mean open times (␥ 1 ϭ 17 pS, 1 ϭ 3.7 msec, and 2 ϭ 23.4 msec; ␥ 2 ϭ 28 pS, 1 ϭ 1.9 msec, and 2 ϭ 8.1 msec). In contrast, mutant AChRs exhibited only one conductance of 11 pS, with 1 ϭ 1.9 msec and 2 ϭ 4.1 msec.The net effect of the mutation is to reduce AChR function. This could result in the hyperexcitability characteristic of epilepsy if the mutant AChRs were part of an inhibitory circuit, e.g., presynaptically regulating the release of GABA. In the minority of AChRs containing the ␣5 subunit, the overall functionality of these AChRs could be maintained despite the mutation in the ␣4 subunit.

The TGF-β signals Nodal, Activin, GDF1, and Vg1 have been implicated in mesoderm induction and left-right patterning. Nodal and Activin both activate Activin receptors, but only Nodal requires EGF-CFC coreceptors for signaling. We report that Vg1 and GDF1 signaling in zebrafish also depends on EGF-CFC proteins, but not on Nodal signals. Correspondingly, we find that inXenopusVg1 and GDF1 bind to and signal through Activin receptors only in the presence of EGF-CFC proteins. These results establish that multiple TGF-β signals converge on Activin receptor/EGF-CFC complexes and suggest a more widespread requirement for coreceptors in TGF-β signaling than anticipated previously.

Polycystin-2 functions as a cation-permeable transient receptor potential ion channel in kidney epithelial cells and when mutated results in human autosomal dominant polycystic kidney disease. For further exploration of the in vivo functions of Polycystin-2, this study examined its expression and function during zebrafish embryogenesis. pkd2 mRNA is ubiquitously expressed, and its presence in the larval kidney could be confirmed by reverse transcription-PCR on isolated pronephroi. Immunostaining with anti-zebrafish Polycystin-2 antibody revealed protein expression in motile kidney epithelial cell cilia and intracellular cell membranes. Intracellular localization was segment specific; in the proximal nephron segment, Polycystin-2 was localized to basolateral cell membranes, whereas in the caudal pronephric segment, Polycystin-2 was concentrated in subapical cytoplasmic vesicles. Polycystin-2 also was expressed in muscle cells and in a variety of sensory cells that are associated with mechanotransduction, including cells of the ear, the lateral line organ, and the olfactory placodes. Disruption of Polycystin-2 mRNA expression resulted in pronephric kidney cysts, body axis curvature, organ laterality defects, and hydrocephalus-defects that could be rescued by expression of a human PKD2 mRNA. In-frame deletions in the first extracellular loop and C-terminal phosphofurin acidic cluster sorting protein-1 (PACS-1) binding sites in the cytoplasmic tail caused Polycystin-2 mislocalization to the apical cell surface. Unlike zebrafish intraflagellar transport protein (IFT) mutants, cyst formation was not associated with cilia defects and instead correlated with reduced kidney fluid output, expansion of caudal duct apical cell membranes, and occlusion of the caudal pronephric nephron segment.