The down-regulation of pank2 gene in zebrafish as a model of Pantothenate Kinase Associated Neurodegeneration.

open9siThe increased iron deposition is a hallmark of many neurodegenerative diseases, but its pathogenic role is still unclear. A strong link between iron and neurodegeneration is evident in a set of heterogeneous neurological disorders, known as Neurodegeneration with Brain Iron Accumulation (NBIA). The most common form of inherited NBIA is associated with mutations in hPank2 gene (PKAN). Pank2 is the rate limiting enzyme in CoA biosynthesis and its downregulation in mammalian cells leads to perturbation of cellular iron homeostasis. Here we explore Pank2 biological function in Danio rerio, and propose this system as an important new tool for the study of PKAN disease.openZizioli, Daniela; Tiso, Natascia; Busolin, Giorgia; Khatri, Deepak; Giuliani, Roberta; Borsani, Giuseppe; Monti, Eugenio; Argenton, Francesco; Finazzi, DarioZizioli, Daniela; Tiso, Natascia; Busolin, Giorgia; Khatri, Deepak; Giuliani, Roberta; Borsani, Giuseppe; Monti, Eugenio; Argenton, Francesco; Finazzi, Dari

Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these compo- nents form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signal- ing, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unex- pected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic pheno- type. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the bal- ance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernu- merary DRG sensory neurons in a wild-type background, and can rescue the sensory neu- ron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10 (baz1) protein\u2019s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lat- eral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates

miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/\u3b2-catenin Signaling Regulation

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/\u3b2-catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease

Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease

Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development

Venous-derived angioblasts generate organ-specific vessels during embryonic development

Formation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. While it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels, as well as the molecular mechanisms regulating their formation, are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas, and as such represents an ideal model to investigate the early steps of organ-specific vessel formation. Here we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the Posterior Cardinal Vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein (SIV) forms and expands ventrally through a bone morphogenetic protein (BMP)-dependent step of collective migration. Concomitantly, a VEGF-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers is observed, which culminates with the generation of the supraintestinal artery (SIA). Altogether our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development, and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late forming-, tissue specific vascular beds

Venous-derived angioblasts generate organ-specific vessels during zebrafish embryonic development

open11siFormation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. Although it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels and the molecular mechanisms regulating their formation are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas and, as such, represents an ideal model in which to investigate the early steps of organ-specific vessel formation. Here, we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the posterior cardinal vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein forms and expands ventrally through a Bone Morphogenetic Protein-dependent step of collective migration. Concomitantly, a Vascular Endothelial Growth Factor-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers, is observed, which culminates with the generation of the supraintestinal artery. Together, our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late-forming, tissue-specific vascular beds.openHen, Gideon; Nicenboim, Julian; Mayseless, Oded; Asaf, Lihee; Shin, Masahiro; Busolin, Giorgia; Hofi, Roy; Almog, Gabriella; Tiso, Natascia; Lawson, Nathan D.; Yaniv, KarinaHen, Gideon; Nicenboim, Julian; Mayseless, Oded; Asaf, Lihee; Shin, Masahiro; Busolin, Giorgia; Hofi, Roy; Almog, Gabriella; Tiso, Natascia; Lawson, Nathan D.; Yaniv, Karin

Transcriptional reprogramming of muscle fibers by chronic electrical stimulation

Background: Skeletal muscle fibers have a remarkable capacity to adjust their molecular, functional, and metabolic properties in response to developmental and environmental stimuli. A central role for neuromuscular activity in determining skeletal muscle fibers composition was demonstrated by cross-innervation and electrical stimulation experiments in adult skeletal muscle. A fast to slow transition can be induced by chronic low-frequency electrical stimulation (CLFS). In literature, CLFS studies have focused on the effects produced after days or weeks of stimulation. Here, I present the first transcriptome study that identifies the earliest genetic changes in this process. Methods: The fast EDL muscle was subjected to CLFS for 6 and 12 hours. First, microarray experiments were performed using whole EDL muscle. Then, in order to reduce biological noise caused by different cell types, I applied microgenomic analyses at the level of single fibers (SF), isolated according to the protocol recently developed in my laboratory. Microarray experiments have been produced with updated platforms (Agilent SurePrint G3 Mouse GE 8x60K). Results and discussion: The expression profiles of whole muscle after 6 and 12 hours of electrical stimulation identified about two hundred differentially expressed (DE) genes. The functional categories of blood vessel development and transcription regulation were most enriched. Genomic analyses of isolated fibers identified more than a thousand DE genes after 12 hours of stimulation. The functional category of blood vessel development was enriched also at the SF level, suggesting that myofibers are able to interact with other cell types in order to stimulate the angiogenesis process. In general, muscle structural genes were equally expressed between stimulated and unstimulated muscles, indicating that changes in fiber type require prolonged stimulation. On the contrary, genes involved in transcription, chromatin-remodeling genes and several myofibril genes acting as signaling molecule were early activated after CLFS.Introduzione: Le fibre del muscolo scheletrico possiedono una notevole capacità di modificare le loro proprietà molecolari, funzionali e metaboliche in risposta a stimoli di crescita e ambientali. Esperimenti di cross-innervazione e stimolazione elettrica hanno dimostrato il ruolo fondamentale dell’attività neuromuscolare nel determinare il tipo di fibra. La stimolazione cronica a bassa frequenza (CLFS) è in grado di indurre il processo di trasformazione di una fibra veloce verso un fenotipo lento. Fino ad ora, questi processi sono stati studiati dopo lunghi periodi di stimolazione. In questo lavoro, mi sono proposta di identificare i cambiamenti trascrizionali precoci di questo processo. Metodi: Il muscolo veloce EDL di topo è stato sottoposto a CLFS per 6 e 12 ore. Inizialmente ho utilizzato il muscolo intero per produrre i profili di espressione Successivamente, per ovviare al problema dell’eterogenea composizione del muscolo, ho utilizzato l’approccio microgenomico, producendo profili di espressione a livello di una singola fibra, isolata utilizzando il protocollo sviluppato nel mio laboratorio. Tutti i profili sono stati ottenuti mediante l’utilizzo di piattaforme Agilent (SurePrint G3 Mouse GE 8x60K). Risultati e discussione: I profili di espressione con il muscolo intero hanno identificato circa 200 geni differenzialmente espressi (DE). Lo sviluppo dei vasi sanguigni e la regolazione della trascrizione sono risultate le categorie funzionali più arricchite. Le analisi genomiche a livello di singola fibra hanno identificato più di mille geni DE dopo 12 ore di stimolazione. La categoria funzionale riguardante lo sviluppo dei vasi sanguigni risulta arricchita anche con questo approccio, suggerendo un’interazione tra le fibre muscolari e gli altri tipi cellulari al fine di stimolare il processo di angiogenesi. La maggiorparte dei geni strutturali risulta essere ugualmente espressa, suggerendo che i cambiamenti nel tipo di fibra richiedono un tempo prolungato. Al contrario, i geni coinvolti nella trascrizione, nel rimodellamento della cromatina e alcuni geni miofibrillari che agiscono come molecule di segnale, si attivano rapidamente in risposta a CLFS

Autosomal dominant essential tremor: a novel family with anticipation

Essential tremor (ET) is a common progressive movement disorder characterized by a clear genetic predisposition. In the last years, many efforts have been done to map susceptibility loci for ET. Here, we report a clinical and genetic study of a family with ET showing autosomal dominant inheritance and anticipation over three generations. The family has five affected members and exhibits a remarkable anticipation of age at onset of the disease along the generations. We excluded linkage to any of the three loci previously mapped in autosomal dominant ET families. Our data suggest the existence of an additional locus in which a repeat expansion is the possible genetic defect underlying ET