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author:("Zhou, weilin")
1.  Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy 
Nature communications  2013;4:1952.
Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca2+ from internal stores to initiate muscle contraction. Defects in EC coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the EC coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca2+ imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 as the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.
doi:10.1038/ncomms2952
PMCID: PMC4056023  PMID: 23736855
2.  Myo1c is an unconventional myosin required for zebrafish glomerular development 
Kidney international  2013;84(6):10.1038/ki.2013.201.
The targeting and organization of podocyte slit diaphragm proteins nephrin and neph1 is critical for development and maintenance of a functional glomerular filtration barrier. Myo1c is a non-muscle myosin motor protein that interacts directly with nephrin and neph1 and mediates their intracellular transport to the podocyte intercellular junction. Here we investigated the necessity of Myo1c in podocyte development using zebrafish as a model system. Immunofluorescence microscopy and in situ RNA hybridization analysis of zebrafish embryos showed that Myo1c is widely expressed in various tissues including the zebrafish glomerulus. Knockdown of the Myo1c gene in zebrafish using antisense morpholino derivatives resulted in an abnormal developmental phenotype that included pericardial edema and dilated renal tubules. Ultra-structural analysis of the glomerulus in Myo1c depleted zebrafish showed abnormal podocyte morphology and absence of the slit diaphragm. Consistent with these observations, the glomerular filter permeability appeared altered in zebrafish in which Myo1c expression was attenuated. The specificity of Myo1c knockdown was confirmed by a rescue experiment in which co-injection of Myo1c morpholino derivatives with orthologous Myo1c mRNA prepared from mouse cDNA lessened phenotypic abnormalities including edema in Myo1c morphants. Thus, our results demonstrate that Myo1c is necessary for podocyte morphogenesis.
doi:10.1038/ki.2013.201
PMCID: PMC3844053  PMID: 23715127
3.  Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy 
Biomedical Optics Express  2014;5(5):1296-1308.
In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.
doi:10.1364/BOE.5.001296
PMCID: PMC4026892  PMID: 24876996
(170.2520) Fluorescence microscopy; (170.6900) Three-dimensional microscopy
4.  Mutation Analysis of 18 Nephronophthisis-associated Ciliopathy Disease Genes using a DNA Pooling and Next-Generation Sequencing Strategy 
Journal of medical genetics  2010;48(2):105-116.
Background
Nephronophthisis-associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity we devised a strategy of DNA pooling with consecutive massively parallel resequencing (MPR).
Methods
In 120 patients with severe NPHP-AC phenotypes we prepared 5 pools of genomic DNA with 24 patients each which were used as templates in order to PCR-amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on a Illumina Genome-Analyzer and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease-based heteroduplex screening and confirmed by Sanger sequencing.
Results
For proof of principle we used DNA from patients with known mutations and demonstrated the detection of 22 out of 24 different alleles (92% sensitivity). MPR led to the molecular diagnosis in 30/120 patients (25%) and we identified 54 pathogenic mutations (27 novel) in 7 different NPHP-AC genes. Additionally, in 24 patients we only found single heterozygous variants of unknown significance.
Conclusions
The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single-gene disorders. The lack of mutations in 75% of patients in our cohort indicates further extensive heterogeneity in NPHP-AC.
doi:10.1136/jmg.2010.082552
PMCID: PMC3913043  PMID: 21068128
Next-generation sequencing; Ciliopathy; Nephronophthisis
5.  ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruption  
The Journal of Clinical Investigation  2013;123(12):5179-5189.
Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q10 (CoQ10) biosynthesis. Mutations in ADCK4 resulted in reduced CoQ10 levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ10 biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ10 treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10.
doi:10.1172/JCI69000
PMCID: PMC3859425  PMID: 24270420
6.  ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling  
The Journal of Clinical Investigation  2013;123(8):3243-3253.
Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.
doi:10.1172/JCI69134
PMCID: PMC3726174  PMID: 23867502
7.  Biomineralization and Size Control of Stable Calcium Phosphate Core Protein Shell Nanoparticles: Potential for Vaccine Applications 
Bioconjugate Chemistry  2012;23(3):610-617.
Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of E. coli Thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50–70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high affinity ACP binding since elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers three weeks post-injection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.
doi:10.1021/bc200654v
PMCID: PMC3310268  PMID: 22263898
8.  Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling 
Chaki, Moumita | Airik, Rannar | Ghosh, Amiya K. | Giles, Rachel H. | Chen, Rui | Slaats, Gisela G. | Wang, Hui | Hurd, Toby W. | Zhou, Weibin | Cluckey, Andrew | Gee, Heon-Yung | Ramaswami, Gokul | Hong, Chen-Jei | Hamilton, Bruce A. | Červenka, Igor | Ganji, Ranjani Sri | Bryja, Vitezslav | Arts, Heleen H. | van Reeuwijk, Jeroen | Oud, Machteld M. | Letteboer, Stef J.F. | Roepman, Ronald | Husson, Hervé | Ibraghimov-Beskrovnaya, Oxana | Ysunaga, Takayuki | Walz, Gerd | Eley, Lorraine | Sayer, John A. | Schermer, Bernhard | Liebau, Max C. | Benzing, Thomas | Le Corre, Stephanie | Drummond, Iain | Joles, Jaap A. | Janssen, Sabine | Allen, Susan J. | Natarajan, Sivakumar | O Toole, John F. | Attanasio, Massimo | Saunier, Sophie | Antignac, Corinne | Koenekoop, Robert K. | Ren, Huanan | Lopez, Irma | Nayir, Ahmet | Stoetzel, Corinne | Dollfus, Helene | Massoudi, Rustin | Gleeson, Joseph G. | Andreoli, Sharon P. | Doherty, Dan G. | Lindstrad, Anna | Golzio, Christelle | Katsanis, Nicholas | Pape, Lars | Abboud, Emad B. | Al-Rajhi, Ali A. | Lewis, Richard A. | Lupski, James R. | Omran, Heymut | Lee, Eva | Wang, Shaohui | Sekiguchi, JoAnn M. | Saunders, Rudel | Johnson, Colin A. | Garner, Elizabeth | Vanselow, Katja | Andersen, Jens S. | Shlomai, Joseph | Nurnberg, Gudrun | Nurnberg, Peter | Levy, Shawn | Smogorzewska, Agata | Otto, Edgar A. | Hildebrandt, Friedhelm
Cell  2012;150(3):533-548.
SUMMARY
Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as ‘ciliopathies’. However, disease mechanisms remain poorly understood. Here we identify by whole exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway, hitherto not implicated in ciliopathies. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164 and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents, and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
doi:10.1016/j.cell.2012.06.028
PMCID: PMC3433835  PMID: 22863007
9.  FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair 
Nature genetics  2012;44(8):910-915.
SUMMARY
Chronic kidney disease (CKD) represents a major health burden1. Its central feature of renal fibrosis is not well understood. By whole exome resequencing in a model disorder for renal fibrosis, nephronophthisis (NPHP), we identified mutations of Fanconi anemia-associated nuclease 1 (FAN1) as causing karyomegalic interstitial nephritis (KIN). Renal histology of KIN is indistinguishable from NPHP except for the presence of karyomegaly2. FAN1 has nuclease activity, acting in DNA interstrand crosslinking (ICL) repair within the Fanconi anemia pathway of DNA damage response (DDR)3–6. We demonstrate that cells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomycin C. However, they do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from patients with Fanconi anemia. We complement ICL sensitivity with wild type FAN1 but not mutant cDNA from individuals with KIN. Depletion of fan1 in zebrafish revealed increased DDR, apoptosis, and kidney cysts akin to NPHP. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms of renal fibrosis and CKD.
doi:10.1038/ng.2347
PMCID: PMC3412140  PMID: 22772369
10.  Aqueous, Protein-Driven Synthesis of Transition Metal-Doped ZnS Immuno-Quantum Dots 
ACS nano  2011;5(10):8013-8018.
The intentional introduction of transition metal impurities in semiconductor nanocrystals is an attractive approach for tuning quantum dot (QD) emission over a wide range of wavelengths. However, the development of effective doping strategies can be challenging, especially if one simultaneously requires a low toxicity crystalline core, a functional protein shell, and a “green”, single-step synthesis process. Here, we describe a simple and environmentally friendly route for the biofabrication of Cu-doped (blue-green) or Mn-doped (yellow-orange) ZnS nanocrystals surrounded by an antibody-binding protein shell. The ZnS:Mn hybrid particles obtained with this method exhibited a 60% enhancement in maximum photoluminescence intensity relative to undoped nanocrystals and have a hydrodynamic diameter inferior to 10 nm. They can be stored for months at 4°C, are stable over a physiological range of pH and salt concentrations, can be decorated with variable amounts of antibodies by direct mixing, and hold promise for biosensing and imaging applications.
doi:10.1021/nn2024896
PMCID: PMC3204801  PMID: 21942544
Quantum dot; Imaging; Molecular Biomimetics; Bionanotechnology
11.  TRPM7 is required within zebrafish sensory neurons for the activation of touch-evoked escape behaviors 
Mutations in the gene encoding TRPM7 (trpm7), a member of the TRP superfamily of cation channels that possesses an enzymatically active kinase at its carboxyl terminus, cause the touch-unresponsive zebrafish mutant touchdown. We identified and characterized a new allele of touchdown, as well as two previously reported alleles, and found that all three alleles harbor mutations which abolish channel activity. Through the selective restoration of TRPM7 expression in sensory neurons we found that TRPM7’s kinase activity, and selectivity for divalent cations over monovalent cations, were dispensable for touch-evoked activation of escape behaviors in zebrafish.
Additional characterization revealed that sensory neurons were present and capable of responding to tactile stimuli in touchdown mutants, indicating that TRPM7 is not required for sensory neuron survival or mechanosensation. Finally, exposure to elevated concentrations of divalent cations was found to restore touch-evoked behaviors in touchdown mutants. Collectively these findings are consistent with a role for zebrafish TRPM7 within sensory neurons in the modulation of neurotransmitter release at central synapses, similar to that proposed for mammalian TRPM7 at peripheral synapses.
doi:10.1523/JNEUROSCI.4950-10.2011
PMCID: PMC3164782  PMID: 21832193
12.  The retinitis pigmentosa protein RP2 interacts with polycystin 2 and regulates cilia-mediated vertebrate development 
Human Molecular Genetics  2010;19(22):4330-4344.
Ciliopathies represent a growing group of human genetic diseases whose etiology lies in defects in ciliogenesis or ciliary function. Given the established entity of renal–retinal ciliopathies, we have been examining the role of cilia-localized proteins mutated in retinitis pigmentosa (RP) in regulating renal ciliogenesis or cilia-dependent signaling cascades. Specifically, this study examines the role of the RP2 gene product with an emphasis on renal and vertebrate development. We demonstrate that in renal epithelia, RP2 localizes to the primary cilium through dual acylation of the amino-terminus. We also show that RP2 forms a calcium-sensitive complex with the autosomal dominant polycystic kidney disease protein polycystin 2. Ablation of RP2 by shRNA promotes swelling of the cilia tip that may be a result of aberrant trafficking of polycystin 2 and other ciliary proteins. Morpholino-mediated repression of RP2 expression in zebrafish results in multiple developmental defects that have been previously associated with ciliary dysfunction, such as hydrocephalus, kidney cysts and situs inversus. Finally, we demonstrate that, in addition to our observed physical interaction between RP2 and polycystin 2, dual morpholino-mediated knockdown of polycystin 2 and RP2 results in enhanced situs inversus, indicating that these two genes also regulate a common developmental process. This work suggests that RP2 may be an important regulator of ciliary function through its association with polycystin 2 and provides evidence of a further link between retinal and renal cilia function.
doi:10.1093/hmg/ddq355
PMCID: PMC2957320  PMID: 20729296
13.  NaV1.6a is required for normal activation of motor circuits normally excited by tactile stimulation 
Developmental neurobiology  2010;70(7):508-522.
A screen for zebrafish motor mutants identified two non-complementing alleles of a recessive mutation that were named non-active (navmi89 and navmi130). nav embryos displayed diminished spontaneous and touch-evoked escape behaviors during the first three days of development. Genetic mapping identified the gene encoding NaV1.6a (scn8aa) as a potential candidate for nav. Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in NaV1.6a that eliminated channel activity when assayed heterologously. Furthermore the injection of RNA encoding wild type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav.
In vivo electrophysiological analysis of the touch-evoked escape circuit indicated that voltage-dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that NaV1.6a was required for touch-induced activation of the swim locomotor network.
doi:10.1002/dneu.20791
PMCID: PMC2900195  PMID: 20225246
zebrafish; NaV1.6; scn8a; motor behaviors
14.  COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness  
The Journal of Clinical Investigation  2011;121(5):2013-2024.
Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10–related forms of SRNS and hearing loss can be molecularly identified and potentially treated.
doi:10.1172/JCI45693
PMCID: PMC3083770  PMID: 21540551
15.  Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy 
Nature genetics  2010;42(10):840-850.
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders featuring dysplasia or degeneration preferentially in kidney, retina, and cerebellum. Here we combine homozygosity mapping with candidate gene analysis by performing “ciliopathy candidate exome capture” followed by massively-parallel sequencing. We detect 12 different truncating mutations of SDCCAG8 in 10 NPHP-RC families. We demonstrate that SDCCAG8 is localized at both centrioles and directly interacts with NPHP-RC-associated OFD1. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in 3D renal cell cultures. This work identifies SDCCAG8 loss of function as a novel cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
doi:10.1038/ng.662
PMCID: PMC2947620  PMID: 20835237
16.  Identification of 11 Novel Mutations in 8 BBS Genes by High-Resolution Homozygosity Mapping 
Journal of medical genetics  2009;47(4):262-267.
Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive disorder characterized by the five cardinal features retinitis pigmentosa, postaxial polydactyly, mental retardation, obesity and hypogenitalism. In addition, renal cysts and other anomalies of the kidney and urinary tract can be present. To date, mutations in 12 BBS genes as well as in MKS1 and CEP290 have been identified as causing BBS. The vast genetic heterogeneity of BBS renders molecular genetic diagnosis difficult in terms of both the time and cost required to screen all 204 coding exons. Here, we report the use of genome-wide homozygosity mapping as a tool to identify homozygous segments at known BBS loci in BBS individuals from inbred and outbred background. In a worldwide cohort of 45 families, we identified, via direct exon sequencing, causative homozygous mutations in 20 families. Eleven of these mutations were novel, thereby increasing the number of known BBS mutations by 5% (11/218). Thus, in the presence of extreme genetic locus heterogeneity, homozygosity mapping provides a valuable approach to the molecular genetic diagnosis of BBS and will facilitate the discovery of novel pathogenic mutations.
doi:10.1136/jmg.2009.071365
PMCID: PMC3017466  PMID: 19797195
Molecular Genetics
17.  touché is required for touch evoked generator potentials within vertebrate sensory neurons 
The process by which light-touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light-touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing ChannelRhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation.
To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP positive peripheral neurites. In wild type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that generator potentials underlie responsiveness to light-touch in zebrafish.
doi:10.1523/JNEUROSCI.1639-10.2010
PMCID: PMC2921932  PMID: 20631165
Somatosensory; Mechanosensory; Touch; Mutant; Nociception; Patch Clamp
18.  Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos 
Autophagy  2009;5(4):520-526.
Autophagy mediates the bulk turnover of cytoplasmic constituents in lysosomes. During embryonic development in animals, a dramatic degradation of yolk proteins and synthesis of zygotic proteins takes place, leading to intracellular remodeling and cellular differentiation. Zebrafish represents a unique system to study autophagy due in part to its rapid embryonic development relative to other vertebrates. The technical advantages of this organism make it uniquely suited to various studies including high-throughput drug screens. To study autophagy in zebrafish, we identified two zebrafish Atg8 homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. Similar to yeast Atg8 and mammalian LC3, zebrafish Lc3 undergoes post-translational modification starting at the pharyngula stage during embryonic development. We observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. In addition, zebrafish Gabarap accumulates within lysosomes upon autophagy induction. Thus, we established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.
PMCID: PMC2754832  PMID: 19221467
embryogenesis; lysosome; LysoTracker; protein targeting; stress
20.  Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy  
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
doi:10.1172/JCI40076
PMCID: PMC2827951  PMID: 20179356
21.  A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations 
PLoS Genetics  2009;5(1):e1000353.
The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.
Author Summary
Many childhood diseases are caused by single-gene mutations of recessive genes, in which a child has inherited one mutated gene copy from each parent causing disease in the child, but not in the parents who are healthy heterozygous carriers. As the two mutations represent the disease cause, gene mapping helped understand disease mechanisms. “Homozygosity mapping” has been particularly useful. It assumes that the parents are related and that a disease-causing mutation together with a chromosomal segment of identical markers (i.e., homozygous markers) is transmitted to the affected child through the paternal and the maternal line from an ancestor common to both parents. Homozygosity mapping seeks out those homozygous regions to map the disease-causing gene. Homozygosity mapping requires families, in which the parents are knowingly related, and have multiple affected children. To overcome these limitations, we applied homozygosity mapping to single affected individuals from outbred populations. In 72 individuals with known homozygous mutations in 13 different recessive disease genes, we performed homozygosity mapping. In 93% we detected the causative gene in a segment of homozygosity. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations. This will strongly accelerate the discovery of novel recessive disease genes.
doi:10.1371/journal.pgen.1000353
PMCID: PMC2621355  PMID: 19165332

Results 1-21 (21)