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1.
Nat Commun ; 13(1): 1282, 2022 03 11.
Article in English | MEDLINE | ID: mdl-35277505

ABSTRACT

Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on samples enriched for isolated OSs show that Bbs1 is required for BBSome-complex stability and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a role for Bbs1/BBSome in OS lipid homeostasis, suggesting a pathomechanism underlying retinal degeneration in BBS.


Subject(s)
Bardet-Biedl Syndrome , Animals , Bardet-Biedl Syndrome/genetics , Cilia/metabolism , Lipids , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Zebrafish/metabolism
2.
EMBO Mol Med ; 12(7): e11861, 2020 07 07.
Article in English | MEDLINE | ID: mdl-32500975

ABSTRACT

The ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development.


Subject(s)
ATPases Associated with Diverse Cellular Activities/genetics , Cataract/genetics , Deafness/genetics , Mutation , Proteasome Endopeptidase Complex/genetics , Proteolysis , Stress, Physiological , Zebrafish Proteins/genetics , Adolescent , Animals , Cataract/pathology , Child , Child, Preschool , Consanguinity , Deafness/physiopathology , Female , Humans , Infant , Male , Nuclear Respiratory Factor 1/genetics , Pedigree , Phenotype , Proteasome Inhibitors/pharmacology , Proteolysis/drug effects , Stress, Physiological/drug effects , Stress, Physiological/genetics , Syndrome , Ubiquitin/metabolism , Zebrafish/genetics
3.
Hum Mutat ; 41(1): 240-254, 2020 01.
Article in English | MEDLINE | ID: mdl-31549751

ABSTRACT

Polydactyly is one of the most frequent inherited defects of the limbs characterized by supernumerary digits and high-genetic heterogeneity. Among the many genes involved, either in isolated or syndromic forms, eight have been implicated in postaxial polydactyly (PAP). Among those, IQCE has been recently identified in a single consanguineous family. Using whole-exome sequencing in patients with uncharacterized ciliopathies, including PAP, we identified three families with biallelic pathogenic variations in IQCE. Interestingly, the c.895_904del (p.Val301Serfs*8) was found in all families without sharing a common haplotype, suggesting a recurrent mechanism. Moreover, in two families, the systemic phenotype could be explained by additional pathogenic variants in known genes (TULP1, ATP6V1B1). RNA expression analysis on patients' fibroblasts confirms that the dysfunction of IQCE leads to the dysregulation of genes associated with the hedgehog-signaling pathway, and zebrafish experiments demonstrate a full spectrum of phenotypes linked to defective cilia: Body curvature, kidney cysts, left-right asymmetry, misdirected cilia in the pronephric duct, and retinal defects. In conclusion, we identified three additional families confirming IQCE as a nonsyndromic PAP gene. Our data emphasize the importance of taking into account the complete set of variations of each individual, as each clinical presentation could finally be explained by multiple genes.


Subject(s)
Ciliopathies/diagnosis , Ciliopathies/genetics , Fingers/abnormalities , Genetic Predisposition to Disease , Genetic Variation , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Phenotype , Polydactyly/diagnosis , Polydactyly/genetics , Toes/abnormalities , Animals , Consanguinity , Fluorescent Antibody Technique , Gene Expression Profiling , Genetic Association Studies/methods , Homozygote , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Pedigree , Signal Transduction , Transcriptome , Exome Sequencing , Zebrafish
4.
PLoS One ; 13(11): e0207747, 2018.
Article in English | MEDLINE | ID: mdl-30458023

ABSTRACT

In zebrafish, the gene choline acetyltransferase a (chata) encodes one of the two ChAT orthologs responsible for the synthesis of acetylcholine. Acetylcholine (ACh) is essential for neuromuscular transmission and its impaired synthesis by ChAT can lead to neuromuscular junction disorders such as congenital myasthenic syndromes in humans. We have identified a novel mutation in the chata gene of zebrafish, chatatk64, in a collection of uncharacterised ENU-induced mutants. This mutant carries a missense mutation in the codon of a highly conserved serine changing it to an arginine (S102R). This serine is conserved among ChATs from zebrafish, rat, mice and chicken to humans. It resides within the catalytic domain and in the vicinity of the active site of the enzyme. However, it has not been reported so far to be required for enzymatic activity. Modelling of the S102R variant change in the ChAT protein crystal structure suggests that the change affects protein structure and has a direct impact on the catalytic domain of the protein which abolishes embryo motility almost completely.


Subject(s)
Catalytic Domain , Choline O-Acetyltransferase/chemistry , Choline O-Acetyltransferase/genetics , Embryo, Nonmammalian/physiology , Movement , Serine , Zebrafish/embryology , Alleles , Amino Acid Sequence , Amino Acid Substitution , Animals , Choline O-Acetyltransferase/metabolism , Enzyme Stability , Humans , Mutation, Missense , Protein Structure, Secondary , Zebrafish/genetics
5.
Biochem Biophys Res Commun ; 496(2): 339-345, 2018 02 05.
Article in English | MEDLINE | ID: mdl-29331378

ABSTRACT

Sarcomeric protein turnover needs to be tightly balanced to assure proper assembly and renewal of sarcomeric units within muscle tissues. The mechanisms regulating these fundamental processes are only poorly understood, but of great clinical importance since many cardiac and skeletal muscle diseases are associated with defective sarcomeric organization. The SET- and MYND domain containing protein 1b (Smyd1b) is known to play a crucial role in myofibrillogenesis by functionally interacting with the myosin chaperones Unc45b and Hsp90α1. In zebrafish, Smyd1b, Unc45b and Hsp90α1 are part of the misfolded myosin response (MMR), a regulatory transcriptional response that is activated by disturbed myosin homeostasis. Genome duplication in zebrafish led to a second smyd1 gene, termed smyd1a. Morpholino- and CRISPR/Cas9-mediated knockdown of smyd1a led to significant perturbations in sarcomere structure resulting in decreased cardiac as well as skeletal muscle function. Similar to Smyd1b, we found Smyd1a to localize to the sarcomeric M-band in skeletal and cardiac muscles. Overexpression of smyd1a efficiently compensated for the loss of Smyd1b in flatline (fla) mutant zebrafish embryos, rescued the myopathic phenotype and suppressed the MMR in Smyd1b-deficient embryos, suggesting overlapping functions of both Smyd1 paralogs. Interestingly, Smyd1a is not transcriptionally activated in Smyd1b-deficient fla mutants, demonstrating lack of genetic compensation despite the functional redundancy of both zebrafish Smyd1 paralogs.


Subject(s)
Gene Expression Regulation, Developmental , Histone-Lysine N-Methyltransferase/genetics , Muscle, Skeletal/metabolism , Myocytes, Cardiac/metabolism , Myosins/genetics , Sarcomeres/metabolism , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Animals, Genetically Modified , CRISPR-Cas Systems , Embryo, Nonmammalian , Gene Duplication , Gene Editing , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/deficiency , Humans , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Morpholinos/genetics , Morpholinos/metabolism , Muscle Proteins , Muscle, Skeletal/pathology , Myocytes, Cardiac/pathology , Myosins/metabolism , Protein Folding , Protein Isoforms/deficiency , Protein Isoforms/genetics , Sarcomeres/pathology , Zebrafish/growth & development , Zebrafish/metabolism , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/deficiency , Zebrafish Proteins/metabolism
6.
Zebrafish ; 14(6): 586-588, 2017 12.
Article in English | MEDLINE | ID: mdl-28767326

ABSTRACT

A bottleneck in CRISPR/Cas9 genome editing is variable efficiencies of in silico-designed gRNAs. We evaluated the sensitivity of the TIDE method (Tracking of Indels by DEcomposition) introduced by Brinkman et al. in 2014 for assessing the cutting efficiencies of gRNAs in zebrafish. We show that this simple method, which involves bulk polymerase chain reaction amplification and Sanger sequencing, is highly effective in tracking well-performing gRNAs in pools of genomic DNA derived from injected embryos. The method is equally effective for tracing INDELs in heterozygotes.


Subject(s)
Genetic Techniques/veterinary , INDEL Mutation , RNA, Guide, Kinetoplastida/genetics , Zebrafish/genetics , Animals , CRISPR-Cas Systems , Gene Editing , Polymerase Chain Reaction/methods , Sequence Analysis, RNA/methods
7.
Nat Commun ; 7: 13586, 2016 11 24.
Article in English | MEDLINE | ID: mdl-27882921

ABSTRACT

Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium.


Subject(s)
Carrier Proteins/metabolism , Cilia/metabolism , Ciliopathies/genetics , Golgi Apparatus/metabolism , Vacuolar Sorting Protein VPS15/genetics , Abnormalities, Multiple/genetics , Adolescent , Animals , Case-Control Studies , Cells, Cultured , Child , Child, Preschool , Craniofacial Abnormalities/complications , Craniofacial Abnormalities/genetics , Female , Fibroblasts/metabolism , Hand Deformities, Congenital/complications , Hand Deformities, Congenital/genetics , Humans , Learning Disabilities/complications , Learning Disabilities/genetics , Male , Mutation , Mutation, Missense , Renal Insufficiency/complications , Renal Insufficiency/genetics , Retinitis Pigmentosa/complications , Retinitis Pigmentosa/genetics , Saccharomyces cerevisiae , Siblings , Skin/cytology , Young Adult , Zebrafish
8.
Data Brief ; 8: 61-8, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27274534

ABSTRACT

Formation of the contractile myofibril of the skeletal muscle is a complex process which when perturbed leads to muscular dystrophy. Herein, we provide a mRNAseq dataset on three different zebrafish mutants affecting muscle organization during embryogenesis. These comprise the myosin folding chaperone unc45b (unc45b-/-), heat shock protein 90aa1.1 (hsp90aa1.1-/-) and the acetylcholine esterase (ache-/-) gene. The transcriptome analysis was performed in duplicate experiments at 72 h post-fertilization (hpf) for all three mutants, with two additional times of development (24 hpf and 48 hpf) for unc45b-/-. A total of 20 samples were analyzed by hierarchical clustering for differential gene expression. The data from this study support the observation made in Etard et al. (2015) [1] (http://dx.doi.org/10.1186/s13059-015-0825-8) that a failure to fold myosin activates a unique transcriptional program in the skeletal muscles that is different from that induced in stressed muscle cells.

9.
Genesis ; 54(8): 431-8, 2016 08.
Article in English | MEDLINE | ID: mdl-27295336

ABSTRACT

Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off-target effects the therapeutic gene should be driven by a tissue-specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue-specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle-specific expression but presents heat-shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b-deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle-specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431-438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.


Subject(s)
Muscle, Skeletal/metabolism , Myocardium/metabolism , Promoter Regions, Genetic , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Muscle Proteins/genetics , Muscle Proteins/metabolism , Organ Specificity , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish
10.
Sci Rep ; 6: 25046, 2016 05 04.
Article in English | MEDLINE | ID: mdl-27141993

ABSTRACT

Altered levels of trace elements are associated with increased oxidative stress that is eventually responsible for pathologic conditions. Oxidative stress has been proposed to be involved in eye diseases, including cataract formation. We visualized the distribution of metals and other trace elements in the eye of zebrafish embryos by micro X-ray fluorescence (µ-XRF) imaging. Many elements showed highest accumulation in the retinal pigment epithelium (RPE) of the zebrafish embryo. Knockdown of the zebrafish brown locus homologues tyrp1a/b eliminated accumulation of these elements in the RPE, indicating that they are bound by mature melanosomes. Furthermore, albino (slc45a2) mutants, which completely lack melanosomes, developed abnormal lens reflections similar to the congenital cataract caused by mutation of the myosin chaperon Unc45b, and an in situ spin trapping assay revealed increased oxidative stress in the lens of albino mutants. Finally transplanting a wildtype lens into an albino mutant background resulted in cataract formation. These data suggest that melanosomes in pigment epithelial cells protect the lens from oxidative stress during embryonic development, likely by buffering trace elements.


Subject(s)
Embryonic Development , Lens, Crystalline/embryology , Lens, Crystalline/physiology , Melanosomes/metabolism , Zebrafish/embryology , Animals , Cataract/prevention & control , Lens, Crystalline/chemistry , Oxidative Stress , Pigments, Biological/metabolism , Spectrometry, X-Ray Emission , Trace Elements/analysis
11.
Genome Biol ; 16: 267, 2015 Dec 03.
Article in English | MEDLINE | ID: mdl-26631063

ABSTRACT

BACKGROUND: Mutations in myosin chaperones Unc45b and Hsp90aa1.1 as well as in the Unc45b-binding protein Smyd1b impair formation of myofibrils in skeletal muscle and lead to the accumulation of misfolded myosin. The concomitant transcriptional response involves up-regulation of the three genes encoding these proteins, as well as genes involved in muscle development. The transcriptional up-regulation of unc45b, hsp90aa1.1 and smyd1b is specific to zebrafish mutants with myosin folding defects, and is not triggered in other zebrafish myopathy models. RESULTS: By dissecting the promoter of unc45b, we identify a Heat shock factor 1 (Hsf1) binding element as a mediator of unc45b up-regulation in myofibers lacking myosin folding proteins. Loss-of-function of Hsf1 abolishes unc45b up-regulation in mutants with defects in myosin folding. CONCLUSIONS: Taken together, our data show that skeletal muscle cells respond to defective myosin chaperones with a complex gene program and suggest that this response is mediated by Hsf1 activation.


Subject(s)
HSP90 Heat-Shock Proteins/genetics , Histone-Lysine N-Methyltransferase/genetics , Molecular Chaperones/genetics , Muscular Diseases/genetics , Myosins/genetics , Zebrafish Proteins/genetics , Animals , Disease Models, Animal , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , HSP90 Heat-Shock Proteins/biosynthesis , Histone-Lysine N-Methyltransferase/biosynthesis , Humans , Molecular Chaperones/biosynthesis , Muscle Proteins , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolism , Muscular Diseases/metabolism , Muscular Diseases/pathology , Mutation , Myosins/metabolism , Protein Binding , Protein Folding , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/biosynthesis
12.
Am J Hum Genet ; 96(4): 666-74, 2015 Apr 02.
Article in English | MEDLINE | ID: mdl-25817018

ABSTRACT

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.


Subject(s)
Choroid Diseases/genetics , Eye Diseases, Hereditary/genetics , Microcephaly/genetics , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Retinal Diseases/genetics , Tubulin/metabolism , Base Sequence , Exome/genetics , Frameshift Mutation/genetics , France , Gene Components , Humans , Microtubules/metabolism , Molecular Sequence Data , Pedigree , Sequence Analysis, DNA
13.
J Med Genet ; 51(2): 132-6, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24026985

ABSTRACT

BACKGROUND: Bardet-Biedl syndrome (BBS) is a recessive and genetically heterogeneous ciliopathy characterised by retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioural dysfunction and hypogonadism. 7 of the 17 BBS gene products identified to date assemble together with the protein BBIP1/BBIP10 into the BBSome, a protein complex that ferries signalling receptors to and from cilia. METHODS AND RESULTS: Exome sequencing performed on a sporadic BBS case revealed for the first time a homozygous stop mutation (NM_001195306: c.173T>G, p.Leu58*) in the BBIP1 gene. This mutation is pathogenic since no BBIP1 protein could be detected in fibroblasts from the patient, and BBIP1[Leu58*] is unable to associate with the BBSome subunit BBS4. CONCLUSIONS: These findings identify BBIP1 as the 18th BBS gene (BBS18) and suggest that BBSome assembly may represent a unifying pathomechanism for BBS.


Subject(s)
Bardet-Biedl Syndrome/genetics , Carrier Proteins/genetics , Codon, Nonsense , Exome , Animals , Bardet-Biedl Syndrome/metabolism , Base Sequence , Consanguinity , DNA Mutational Analysis , Fibroblasts/metabolism , Genetic Association Studies , Genetic Linkage , HEK293 Cells , Humans , Male , Middle Aged , Molecular Sequence Annotation , Pedigree , Phenotype , Polymorphism, Single Nucleotide , Zebrafish
14.
Proc Natl Acad Sci U S A ; 110(47): 18982-7, 2013 Nov 19.
Article in English | MEDLINE | ID: mdl-24191061

ABSTRACT

Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.


Subject(s)
Drosophila/embryology , Gene Expression Regulation, Developmental/physiology , Giant Cells/physiology , Glycolysis/physiology , Muscles/embryology , Myoblasts/physiology , Animals , Cell Fusion , Gene Expression Regulation, Developmental/genetics , Glycolysis/genetics , In Situ Hybridization , Insulin/metabolism , Pyruvate Kinase/metabolism , RNA Interference , Statistics, Nonparametric , Zebrafish
15.
Am J Hum Genet ; 89(6): 773-81, 2011 Dec 09.
Article in English | MEDLINE | ID: mdl-22152679

ABSTRACT

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on a severe developmental dental defect that results in a dentin dysplasia phenotype with major microdontia, oligodontia, and shape abnormalities in a highly consanguineous family. Homozygosity mapping revealed a unique zone on 6q27-ter. The two affected children were found to carry a homozygous mutation in SMOC2. Knockdown of smoc2 in zebrafish showed pharyngeal teeth that had abnormalities reminiscent of the human phenotype. Moreover, smoc2 depletion in zebrafish affected the expression of three major odontogenesis genes: dlx2, bmp2, and pitx2.


Subject(s)
Calcium-Binding Proteins/genetics , Chromosome Mapping , Dentin Dysplasia/genetics , Exome , Homozygote , Sequence Analysis, DNA , Tooth/growth & development , Adaptor Proteins, Signal Transducing , Animals , Carrier Proteins/genetics , Child , Child, Preschool , Chromosomes, Human, Pair 6 , Consanguinity , Dentin Dysplasia/diagnosis , Female , Gene Expression , Gene Expression Regulation, Developmental , Genetic Association Studies , Humans , Mice , Molecular Sequence Data , Neoplasm Proteins/genetics , Pedigree , Zebrafish/genetics , Zebrafish/growth & development
16.
J Cell Sci ; 124(Pt 18): 3127-36, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21852424

ABSTRACT

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.


Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Muscle, Skeletal/enzymology , Myocardium/enzymology , Myosins/metabolism , Sarcomeres/metabolism , Zebrafish Proteins/metabolism , Animals , Cloning, Molecular , Cytoskeleton/metabolism , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/genetics , Microarray Analysis , Muscle Contraction/physiology , Muscle, Skeletal/ultrastructure , Mutation/genetics , Myocardium/ultrastructure , Protein Binding , Sarcomeres/genetics , Transgenes/genetics , Zebrafish , Zebrafish Proteins/genetics
17.
J Cell Biol ; 189(3): 527-39, 2010 May 03.
Article in English | MEDLINE | ID: mdl-20440001

ABSTRACT

The chaperones Unc45b and Hsp90a are essential for folding of myosin in organisms ranging from worms to humans. We show here that zebrafish Unc45b, but not Hsp90a, binds to the putative cytidine deaminase Apobec2 (Apo2) in an interaction that requires the Unc45/Cro1p/She4p-related (UCS) and central domains of Unc45b. Morpholino oligonucleotide-mediated knockdown of the two related proteins Apo2a and Apo2b causes a dystrophic phenotype in the zebrafish skeletal musculature and impairs heart function. These phenotypic traits are shared with mutants of unc45b, but not with hsp90a mutants. Apo2a and -2b act nonredundantly and bind to each other in vitro, which suggests a heteromeric functional complex. Our results demonstrate that Unc45b and Apo2 proteins act in a Hsp90a-independent pathway that is required for integrity of the myosepta and myofiber attachment. Because the only known function of Unc45b is that of a chaperone, Apo2 proteins may be clients of Unc45b but other yet unidentified processes cannot be excluded.


Subject(s)
Cytidine Deaminase/genetics , Embryo, Nonmammalian/metabolism , Muscle, Skeletal/embryology , Phenotype , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Cytidine Deaminase/metabolism , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Muscle Proteins , Muscle, Skeletal/metabolism , Type C Phospholipases/genetics , Type C Phospholipases/metabolism , Zebrafish/metabolism , Zebrafish Proteins/metabolism
18.
J Cell Biol ; 180(6): 1163-75, 2008 Mar 24.
Article in English | MEDLINE | ID: mdl-18347070

ABSTRACT

The formation of thick filaments in striated muscle involves the chaperones Hsp90a and Unc45. We show that Unc45b and Hsp90a, two zebrafish orthologues, colocalize with myosin during myofibrillogenesis and associate with the Z line when myofibril assembly is completed. In response to stress or damage to the myofiber, Unc45b and Hsp90a dissociate from the Z line and transiently associate with myosin. Although chaperone activity of Unc45b requires the full-length protein, only the central and Unc45-Cro1p-She4p domains are required to anchor it to the Z line, and multiple subdomains mediate association with nascent myosin. We propose that the Z line serves as a reservoir for chaperones, allowing a rapid mobilization in response to muscle damage. Our data are consistent with a differential affinity model as an explanation for the shuttling of the chaperones between the Z line and myosin.


Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Intracellular Membranes/metabolism , Molecular Chaperones/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle, Striated/metabolism , Zebrafish Proteins/metabolism , Animals , Intracellular Membranes/ultrastructure , Models, Biological , Muscle Fibers, Skeletal/ultrastructure , Muscle Proteins , Muscle, Striated/injuries , Muscle, Striated/ultrastructure , Myosins/metabolism , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Protein Transport/physiology , Regeneration/physiology , Stress, Mechanical , Time Factors , Zebrafish
19.
Development ; 135(6): 1147-56, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18256191

ABSTRACT

The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the zebrafish sloth(u45) mutant, in which the initial steps in sarcomere assembly take place, but thick filaments are absent and filamentous I-Z-I brushes fail to align or adopt correct spacing. The mutation only affects skeletal muscle and mutant embryos show no other obvious phenotypes. Surprisingly, we find that the phenotype is due to mutation in one copy of a tandemly duplicated hsp90a gene. The mutation disrupts the chaperoning function of Hsp90a through interference with ATPase activity. Despite being located only 2 kb from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to the downregulation of genes encoding sarcomeric proteins and upregulation of hsp90a and several other genes encoding proteins that may act with Hsp90a during sarcomere assembly. Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly.


Subject(s)
Adenosine Triphosphatases/metabolism , HSP90 Heat-Shock Proteins/metabolism , Muscle Development/physiology , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolism , Zebrafish Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/deficiency , Adenosine Triphosphatases/genetics , Animals , Base Sequence , Binding Sites , DNA Primers/genetics , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/deficiency , HSP90 Heat-Shock Proteins/genetics , Heat-Shock Response , Microscopy, Electron, Transmission , Models, Molecular , Mutation , Myofibrils/metabolism , Phenotype , Sarcomeres/metabolism , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish/metabolism , Zebrafish Proteins/chemistry , Zebrafish Proteins/deficiency , Zebrafish Proteins/genetics
20.
Dev Biol ; 308(1): 133-43, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17586488

ABSTRACT

Contraction of muscles is mediated by highly organized arrays of myosin motor proteins. We report here the characterization of a mutation of a UCS gene named steif/unc-45b that is required for the formation of ordered myofibrils in both the skeletal and cardiac muscles of zebrafish. We show that Steif/Unc-45b interacts with the chaperone Hsp90a in vitro. The two genes are co-expressed in the skeletal musculature and knockdown of Hsp90a leads to impaired myofibril formation in the same manner as lack of Steif/Unc-45b activity. Transcripts of both genes are up-regulated in steif mutants suggesting co-regulation of the two genes. Our data indicate a requirement of Steif/unc-45b and Hsp90a for the assembly of the contractile apparatus in the vertebrate skeletal musculature.


Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Muscle Development/physiology , Muscle Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , DNA/genetics , Gene Expression Regulation, Developmental , HSP90 Heat-Shock Proteins/genetics , Heart/embryology , In Vitro Techniques , Microscopy, Electron, Transmission , Molecular Sequence Data , Muscle Development/genetics , Muscle Proteins/genetics , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Muscle, Skeletal/ultrastructure , Mutation , Myocardium/metabolism , Myocardium/ultrastructure , Phenotype , Zebrafish/genetics , Zebrafish Proteins/genetics
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