Search

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, yet the genetic cause of up to 50% of cases remains unknown. Here, we show that mutations in KLHL24 cause HCM in humans. Using genome-wide linkage analysis and exome sequencing, we identified homozygous mutations in KLHL24 in two consanguineous families with HCM. Of the 11 young affected adults identified, 3 died suddenly and 1 had a cardiac transplant due to heart failure. KLHL24 is a member of the Kelch-like protein family, which acts as substrate-specific adaptors to Cullin E3 ubiquitin ligases. Endomyocardial and skeletal muscle biopsies from affected individuals of both families demonstrated characteristic alterations, including accumulation of desmin intermediate filaments. Knock-down of the zebrafish homologue klhl24a results in heart defects similar to that described for other HCM-linked genes providing additional support for KLHL24 as a HCM-associated gene. Our findings reveal a crucial role for KLHL24 in cardiac development and function.

Skeletal muscle derives from dorsal mesoderm formed during vertebrate gastrulation. Fibroblast growth factor (Fgf) signalling cooperates with Tbx transcription factors to promote dorsal mesoderm formation, but their role in myogenesis has been unclear. Using zebrafish, we show that dorsally derived Fgf signals act through Tbx16 and Tbxta to induce slow and fast trunk muscle precursors at distinct dorsoventral positions. Tbx16 binds to and directly activates the myf5 and myod genes, which are required for commitment to myogenesis. Tbx16 activity depends on Fgf signalling from the organiser. In contrast, Tbxta is not required for myf5 expression, but binds a specific site upstream of myod that is not bound by Tbx16 and drives (dependent on Fgf signals) myod expression in adaxial slow precursors, thereby initiating trunk myogenesis. After gastrulation, when similar muscle cell populations in the post-anal tail are generated from tailbud, declining Fgf signalling is less effective at initiating adaxial myogenesis, which is instead initiated by Hedgehog signalling from the notochord. Our findings suggest a hypothesis for ancestral vertebrate trunk myogenic patterning and how it was co-opted during tail evolution to generate similar muscle by new mechanisms. This article has an associated ‘The people behind the papers’ interview., Highlighted Article: Tbx16 and Tbxta activate myf5 and myod directly during the earliest myogenesis in zebrafish, and Fgf signalling acts through Tbx16 to drive myogenesis in trunk but not tail.

With the advances in next-generation sequencing and rapid filtering of candidate variants in diseased patients, it has been increasingly important to develop translatable in vivo models to study genetic changes. This allows for functional validation of pathogenic mutations and establishes a system to understand the etiology of disease. Due to the ease of genetic manipulation and rapid ex utero development, the zebrafish has become a valuable resource to study important biological processes, including nephrogenesis. The development and function of the zebrafish pronephros are akin to that of mammals. As such, they offer a tractable model to study kidney disease, especially diabetic nephropathy. However, in order to study kidney dysfunction in zebrafish it is imperative that an appropriate readout is available. The appearance of macro-proteins in patient's urine is indicative of defective kidney function. In this technical chapter, we describe the in vivo use of fluorescently tagged dextrans of different molecular weights to reveal the integrity of the zebrafish glomerular filtration barrier.

Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade body (WPB) exocytosis and secretion of Von Willebrand factor (VWF) from endothelial cells, however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remain unknown. Here we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVEC) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVEC identified SYT5 protein, and exogenously expressed SYT5-mEGFP localized almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

OBJECTIVE: Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, approximately 50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. METHODS: We performed exome sequencing on two consanguineous probands diagnosed with a congenital myopathy and muscle biopsy showing selective atrophy/hypotrophy or absence of type II myofibres. RESULTS: We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain in both probands. A homozygous essential splice acceptor variant (c.479-2A>G, predicted to result in skipping of exon 5 was identified in Proband 1, and a homozygous missense substitution (c.488T>G, p.(Met163Arg)) was identified in Proband 2. Protein modeling of the p.(Met163Arg) substitution predicted it might impede intermolecular interactions that facilitate binding to the IQ domain of myosin heavy chain, thus likely impacting on the structure and functioning of the myosin motor. MYL1 was markedly reduced in skeletal muscle from both probands, suggesting that the missense substitution likely results in an unstable protein. Knock down of myl1 in zebrafish resulted in abnormal morphology, disrupted muscle structure and impaired touch-evoked escape responses, thus confirming that skeletal muscle fast-twitch specific myosin essential light chain is critical for myofibre development and function. INTERPRETATION: Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with a severe congenital myopathy.

Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic causes of IS are poorly understood. Turner syndrome (TS) is caused by haploinsufficiency for a subset of genes within the pseudoautosomal region (PAR) at the tip of the short arm of the X chromosome thereby defining a critical interval for TS pathogenesis. Patients with TS present with a number of clinical features that individually represent common diseases, including an increased risk of developing IS. It is therefore important to assign genes within the TS critical interval to each of these disorders. Here, we focus on one gene within this interval, PPP2R3B, which encodes a protein phosphatase 2A regulatory subunit that interacts with the origin of replication component, CDC6, and has been linked to regulation of autophagy. We found that PPP2R3B is expressed at sites of chondrogenesis within human foetuses, including the vertebrae. We also demonstrated prominent expression in myotome and muscle fibres in human foetuses, and zebrafish embryos and adolescents. As there is no rodent orthologue of PPP2R3B, we used CRIPSR/Cas9-mediated gene-editing to generate a frameshift mutation in zebrafish ppp2r3b. Adolescent zebrafish that were homozygous for this mutation exhibited a fully penetrant kyphoscoliosis phenotype which became progressively worse over time, mirroring IS in humans. These defects were associated with reduced mineralisation of vertebrae, resembling osteoporosis. Electron microscopy demonstrated abnormal mitochondria adjacent to muscle fibres, a feature which is associated with muscular dystrophy. Collectively, our data support a role for PPP2R3B haploinsufficiency in human IS pathogenesis in TS, establish a new animal model of this condition and provide a basis to investigate the underlying mechanisms. Author Summary Abnormal curvature of the spine is extremely common, often developing as people age. The causes of this are not well understood and a major focus of active research is to identify the underlying genetic causes of this. In this study, we used gene-editing to create zebrafish carrying a mutation in the gene PPP2R3B. This is a gene linked to Turner syndrome in which patients develop spinal curvature, however, it has never been implicated in skeletal tissue maintenance before. Adolescent mutant zebrafish developed spinal curvature which became progressively worse, mirroring the human condition. This was associated with reduced formation of mineralised bone including an osteoporosis-like presentation within vertebrae. This was associated with prominent ppp2r3b expression in axial muscle and a muscular dystrophy-like phenotype. This work provides an experimental starting point to investigate the molecular and cellular mechanisms underlying spinal tissue integrity in future.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11’s role in development is poorly understood. Here we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues.Wdr11null mice also exhibit early onset obesity. We found that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 was also observed to regulate the proteolytic processing of GLI3 and cooperate with EMX1 transcription factor to induce the expression of downstream Hh pathway genes and gonadotrophin releasing hormone production. The CHH/KS-associated human mutations result in loss-of-function of WDR11. Treatment with the Hh agonist purmorphamine partially rescued the WDR11-haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

We describe a mutation inLMOD1, which predisposes individuals to thoracic aortic aneurysms and dissections in a large multi-generation British family. Exome variant profiles for the proband and two distantly related affected relatives were generated and a rare protein-altering, heterozygous variant was identified, present in all the exome-sequenced affected individuals. The allele c.1784T>C, p.(V595A) inLMOD1is located in a known actin-binding WH2 domain and is carried by all living affected individuals in the family.LMOD1was further assessed in a consecutive series of 98 UK TAAD patients and one further mutation was found, yielding an incidence of ∼2% in our study group. Assessment ofLMOD1in international TAAD cohorts discovered nine other missense variants of which three were classed as likely pathogenic.Validation ofLMOD1was undertaken using a zebrafish animal model. Knock-down of bothlmod1aandlmod1bparalogs using morpholino oligonucleotides showed a reproducible abnormal phenotype involving the aortic arches under off-target controls. Injection of the humanLMOD1c.1784T>C, p.(V595A) mutation demonstrated a likely dominant negative effect and illustrated a loss of function cause.Mutations found in the WH2 actin-binding domain ofLMOD1may delay actin polymerization and therefore compromise actin length, dynamics and interaction with myosin in the smooth muscle contraction pathway.

Congenital muscular dystrophies display a wide phenotypic and genetic heterogeneity. The combination of clinical, biochemical, and molecular genetic findings must be considered to obtain the precise diagnosis and provide appropriate genetic counselling. Here we report five individuals from four families presenting with variable clinical features including muscular dystrophy with a reduction in dystroglycan glycosylation, short stature, intellectual disability, and cataracts, overlapping both the dystroglycanopathies and Marinesco-Sjogren syndrome. Whole-exome sequencing revealed homozygous missense and compound heterozygous mutations in INPP5K in the affected members of each family. INPP5K encodes the inositol polyphosphate-5-phosphatase K, also known as SKIP (skeletal muscle and kidney enriched inositol phosphatase), which is highly expressed in the brain and muscle. INPP5K localizes to both the endoplasmic reticulum and to actin ruffles in the cytoplasm. It has been shown to regulate myoblast differentiation and has also been implicated in protein processing through its interaction with the ER chaperone HSPA5/BiP. We show that morpholino-mediated inpp5k loss of function in the zebrafish results in shortened body axis, microphthalmia with disorganized lens, microcephaly, reduced touch-evoked motility, and highly disorganized myofibers. Altogether these data demonstrate that mutations in INPP5K cause a congenital muscular dystrophy syndrome with short stature, cataracts, and intellectual disability.

Understanding the role of basal bodies (BBs) during development and disease has been largely overshadowed by research into the function of the cilium. Although these two organelles are closely associated, they have specific roles to complete for successful cellular development. Appropriate development and function of the BB are fundamental for cilia function. Indeed, there are a growing number of human genetic diseases affecting ciliary development, known collectively as the ciliopathies. Accumulating evidence suggests that BBs establish cell polarity, direct ciliogenesis, and provide docking sites for proteins required within the ciliary axoneme. Major contributions to our knowledge of BB structure and function have been provided by studies in flagellated or ciliated unicellular eukaryotic organisms, specifically Tetrahymena and Chlamydomonas. Reproducing these and other findings in vertebrates has required animal in vivo models. Zebrafish have fast become one of the primary organisms of choice for modeling vertebrate functional genetics. Rapid ex-utero development, proficient egg laying, ease of genetic manipulation, and affordability make zebrafish an attractive vertebrate research tool. Furthermore, zebrafish share over 80 % of disease causing genes with humans. In this article, we discuss the merits of using zebrafish to study BB functional genetics, review current knowledge of zebrafish BB ultrastructure and mechanisms of function, and consider the outlook for future zebrafish-based BB studies.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that is generally inherited in an autosomal recessive fashion. However, in some families, trans mutant alleles interact with the primary causal locus to modulate the penetrance and/or the expressivity of the phenotype. CCDC28B (MGC1203) was identified as a second site modifier of BBS encoding a protein of unknown function. Here we report the first functional characterization of this protein and show it affects ciliogenesis both in cultured cells and in vivo in zebrafish. Consistent with this biological role, our in silico analysis shows that the presence of CCDC28B homologous sequences is restricted to ciliated metazoa. Depletion of Ccdc28b in zebrafish results in defective ciliogenesis and consequently causes a number of phenotypes that are characteristic of BBS and other ciliopathy mutants including hydrocephalus, left-right axis determination defects and renal function impairment. Thus, this work reports CCDC28B as a novel protein involved in the process of ciliogenesis whilst providing functional insight into the cellular basis of its modifier effect in BBS patients.

3MC syndrome has been proposed as a unifying term encompassing the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability and genital, limb and vesicorenal anomalies. Here we studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP1, both of which encode proteins in the lectin complement pathway (collectin kidney 1 (CL-K1) and MASP-1 and MASP-3, respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney and vertebral bodies. Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities. Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration. Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.

Laterality defects such as situs inversus are not uncommonly encountered in humans, either in isolation or as part of another syndrome, but can have devastating developmental consequences. The events that break symmetry during early embryogenesis are highly conserved amongst vertebrates and involve the establishment of unidirectional flow by cilia within an organising centre such as the node in mammals or Kupffer's vesicle (KV) in teleosts. Disruption of this flow can lead to the failure to successfully establish left-right asymmetry. The correct apical-posterior cellular position of each node/KV cilium is critical for its optimal radial movement which serves to sweep fluid (and morphogens) in the same direction as its neighbours. Planar cell polarity (PCP) is an important conserved process that governs ciliary position and posterior tilt; however the underlying mechanism by which this occurs remains unclear. Here we show that Bbs8, a ciliary/basal body protein important for intraciliary/flagellar transport and the core PCP protein Vangl2 interact and are required for establishment and maintenance of left-right asymmetry during early embryogenesis in zebrafish. We discovered that loss of bbs8 and vangl2 results in laterality defects due to cilia disruption at the KV. We showed that perturbation of cell polarity following abrogation of vangl2 causes nuclear mislocalisation, implying defective centrosome/basal body migration and apical docking. Moreover, upon loss of bbs8 and vangl2, we observed defective actin organisation. These data suggest that bbs8 and vangl2 act synergistically on cell polarization to establish and maintain the appropriate length and number of cilia in the KV and thereby facilitate correct LR asymmetry.

Myogenic regulatory factors of the Myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. However, the roles of each gene in myogenesis remain unclear, owing partly to genetic linkage at the Myf5/Mrf4 locus and to rapid morphogenetic movements in the amniote somite. In mice, Myf5 is essential for the earliest epaxial myogenesis, whereas Myod is required for timely differentiation of hypaxially derived muscle. A second major subdivision of the somite is between primaxial muscle of the somite proper and abaxial somite-derived migratory muscle precursors. Here, we use a combination of mutant and morphant analysis to ablate the function of each of the four conserved MRF genes in zebrafish, an organism that has retained a more ancestral bodyplan. We show that a fundamental distinction in somite myogenesis is into medial versus lateral compartments, which correspond to neither epaxial/hypaxial nor primaxial/abaxial subdivisions. In the medial compartment, Myf5 and/or Myod drive adaxial slow fibre and medial fast fibre differentiation. Myod-driven Myogenin activity alone is sufficient for lateral fast somitic and pectoral fin fibre formation from the lateral compartment, as well as for cranial myogenesis. Myogenin activity is a significant contributor to fast fibre differentiation. Mrf4 does not contribute to early myogenesis in zebrafish. We suggest that the differential use of duplicated MRF paralogues in this novel two-component myogenic system facilitated the diversification of vertebrates.

Poc1 shores up basal bodies to support cilia formation in Tetrahymena thermophila, zebrafish, and humans; Poc1 depletion causes phenotypes commonly seen in ciliopathies., Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis.

The vestigial gene has been shown to control skeletal muscle formation in Drosophila and the related Vestigial-like 2 (Vgl-2) protein plays a similar role in mice. Vgl-family proteins are thought to regulate tissue-specific gene expression by binding to members of the broadly expressed Scalloped/Tef/TEAD transcription factor family. Zebrafish have at least four Vgl genes, including two Vgl-2s, and at least three TEAD genes, including two Tead3s. We describe the cloning and expression of one member from each family in the zebrafish. A novel gene, vgl-2b, with closest homology to mouse and human vgl-2, is expressed transiently in nascent notochord and in muscle fibres as they undergo terminal differentiation during somitogenesis. Muscle cells also express a TEAD-3 homologue, a possible partner of Vgl-2b, during myoblast differentiation and early fibre assembly. Tead-3a is also expressed in rhombomeres, eye and epiphysis regions.

Pax3/7 paired homeodomain transcription factors are important markers of muscle stem cells. Pax3 is required upstream of myod for lateral dermomyotomal cells in the amniote somite to form particular muscle cells. Later Pax3/7-dependent cells generate satellite cells and most body muscle. Here we analyse early myogenesis from, and regulation of, a population of Pax3-expressing dermomyotome-like cells in the zebrafish. Zebrafish pax3 is widely expressed in the lateral somite and, along with pax7, becomes restricted anteriorly and then to the external cells on the lateral somite surface. Midline-derived Hedgehog signals appear to act directly on lateral somite cells to repress Pax3/7. Both Hedgehog and Fgf8, signals that induce muscle formation within the somite, suppress Pax3/7 and promote expression of myogenic regulatory factors (MRFs) myf5 and myod in specific muscle precursor cell populations. Loss of MRF function leads to loss of myogenesis by specific populations of muscle fibres, with parallel up-regulation of Pax3/7. Myod is required for lateral fast muscle differentiation from pax3-expressing cells. In contrast, either Myf5 or Myod is sufficient to promote slow muscle formation from adaxial cells. Thus, myogenic signals act to drive somite cells to a myogenic fate through up-regulation of distinct combinations of MRFs. Our data show that the relationship between Pax3/7 genes and myogenesis is evolutionarily ancient, but that changes in the MRF targets for particular signals contribute to myogenic differences between species.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria ‘dipstick’, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. In this study we investigate the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells cilia loss correlated with a reduction in ligand dependent hedgehog signalling. Heat shock dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by HDAC6 which localises to ciliary axonemes. The rate of cilia resorption was reduced in thermotolerant cells. This implies a role for molecular chaperones in primary cilia maintenance. The cytosolic chaperone Hsp90 localised to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells Hsp90 is known to exist in a complex with HDAC6. Immediately after heat shock Hsp90 levels were reduced in remaining ciliary axonemes. We hypothesise that cilia resorption in response to heat shock is regulated by the disassembly of an HDAC6/Hsp90 complex and would serve to attenuate cilia mediated signalling pathways and reduce the translational load on the cell in times of stress.

Distal arthrogryposis (DA) is a clinically and genetically heterogeneous condition with mutations in at least five genes encoding for sarcomeric proteins (TNN12, TNNT3, TPM2, MYH3 and MYH8) implicated to date. A significant proportion of cases remain genetically uncharacterised. Here we describe 4 members from a 3 generation consanguineous Irish family that presented with a congenital myopathy resembling King-Denborough syndrome (KDS), characterized by clinical features of ptosis and distal arthrogryposis, and cores on muscle biopsy. Through collaboration with The Sanger Centre as part of the UK10K project, we exome screened these subjects and were able to identify a segregating homozygous misssense mutation in exon 2 of the Endothelin converting enzyme like 1 (ECEL1) gene, recently found to be responsible for DA5D. Population screening enabled us to find two additional DA families that harboured causative variants ECEL1 exons 2 and 15, respectively, reinforcing the notion of a causative link between ecel1 dysfunction and the DA’s. ECEL1 encodes for a type2 transmembrane M13 metallopeptidase with an as yet unidentified substrate, localised to the endoplasmic reticulum and plasma membrane of the central nervous system (CNS). The ecel1 protein is also expressed in skeletal muscle and thought to participate in the formation of the neuromuscular junction (NMJ), but its precise function is currently unknown. We therefore embarked on protein function assays in zebrafish morphants and ECEL1-mutated patient fibroblasts. In particular, we knocked down (KD) ecel1 in zebra fish embryo applying a morpholino approach and found a gross defect in body axis curvature leading to perturbed functional abilities. Microscopic examination revealed disorganised/detached myofibres that have intact sarcolemma. (We have also obtained a zebrafish ecel1 knock out (KO) model achieved by insertion of a null allele by chemical mutagenesis. This model does not exhibit the same trunk curvatur.

Differentiation often requires conversion of analogue signals to a stable binary output through positive feedback. Hedgehog (Hh) signalling promotes myogenesis in the vertebrate somite, in part by raising the activity of muscle regulatory factors (MRFs) of the Myod family above a threshold. Hh is known to enhance MRF expression. Here we show that Hh is also essential at a second step that increases Myod protein activity, permitting it to promote Myogenin expression. Hh acts by inducing expression of cdkn1c (p57Kip2) in slow muscle precursor cells, but neither Hh nor Cdkn1c is required for their cell cycle exit. Cdkn1c co-operates with Myod to drive differentiation of several early zebrafish muscle fibre types. Myod in turn up-regulates cdkn1c, thereby providing a positive feedback loop that switches myogenic cells to terminal differentiation.

Mrf4 (Myf6) is a member of the basic helix-loop-helix (bHLH) myogenic regulatory transcription factor (MRF) family, which also contains Myod, Myf5 and myogenin. Mrf4 is implicated in commitment of amniote cells to skeletal myogenesis and is also abundantly expressed in many adult muscle fibres. The specific role of Mrf4 is unclear both because mrf4 null mice are viable, suggesting redundancy with other MRFs, and because of genetic interactions at the complex mrf4lmyf5 locus. We report the cloning and expression of an mrf4 gene from zebrafish, Danio rerio, which shows conservation of linkage to myf5. Mrf4 mRNA accumulates in a subset of terminally differentiated muscle fibres in parallel with myosin protein in the trunk and fin. Although most, possibly all, trunk muscle expresses mrf4, the level of mRNA is dynamically regulated. No expression is detected in muscle precursor cell populations prior to myosin accumulation. Moreover, mrf4 expression is not detected in head muscles, at least at early stages. As fish mature, mrf4 expression is pronounced in the region of slow muscle fibres. (c) 2007 Elsevier B.V. All rights reserved.

Summary Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too.

Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions., Disturbances in IP3 receptor-mediated release of Ca2+ from the endoplasmatic reticulum are associated with neurodegenerative disease. Here, the authors identify in four families with hereditary spastic paraplegia biallelic mutations in RNF170 that associate with increased basal levels of IP3 receptors.

CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet-Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo, at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling.

Polycystin-1 (PKD1) forms a mechanosensitive cation channel complex with polycystin-2 (PKD2) in the primary cilium. Functional defects in this complex caused by mutation of PKD1 and PKD2 result in autosomal dominant polycystic kidney disease (ADPKD). The mechanisms by which this complex regulates normal cell physiology remain elusive. In particular the proteins that interact directly with polycystin-1 remain poorly characterised. A Y-2H screen using the C-terminus of polycystin-1 as bait identified a novel binding partner that we have called PIP9 (polycystin interacting protein 9). PIP9 is evolutionary conserved, ubiquitously expressed, and predicted to be a 9KDa phosphoprotein containing a single coiled-coil domain that interacts with the coiled-coil domain of PC-1. Specific anti-PIP9 antisera confirmed the widespread and developmentally regulated expression of PIP9 and identified phosphorylated isoforms. PIP9 co-localised to the renal primary cilium with PC1 and immuno-EM studies revealed localisation to IFT particles. Knockdown of zPip9, using morpholino oligonucleotides, resulted in developmental defects in gastrulation movements. In addition, evidence of cell detachment was observed, common to the phenotype described for double bbs4;bbs10 morphant zebrafish which suggests pip9 may act in the same genetic pathway. zpip9 morphants also displayed variable head and eye degeneration, including coloboma and hydrocephalus, pronephric cysts and shortening of the body axis. Loss of Pip9 function in mice is lethal before embryonic day E11.5. This preliminary analysis suggests that PIP9 plays a central role in mediating polycystin-1 dependant signalling.