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

Formation 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.

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.

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.

Low penetrance and effect on protein secretion of LGI1 mutations causing autosomal dominant lateral temporal epilepsy.

PURPOSE: To describe the clinical and genetic findings of four families with autosomal dominant lateral temporal epilepsy. METHODS: A personal and family history was obtained from each affected and unaffected subject along with a physical and neurologic examination. Routine electroencephalography and magnetic resonance imaging (MRI) studies were performed in almost all patients. DNAs from family members were screened for LGI1 mutations. The effects of mutations on Lgi1 protein secretion were determined in transfected culture cells. KEY FINDINGS: The four families included a total of 11 patients (two deceased), six of whom had lateral temporal epilepsy with auditory aura. Age at onset was in the second decade of life; seizures were well controlled by antiepileptic treatment and MRI studies were normal. We found two pathogenic LGI1 mutations with uncommonly low penetrance: the R136W mutation, previously detected in a sporadic case with telephone-induced partial seizures, gave rise to the epileptic phenotype in three of nine mutation carriers in one family; the novel C179R mutation caused epilepsy in an isolated patient from a family where the mutation segregated. Another novel pathogenic mutation, I122T, and a nonsynonymous variant, I359V, were found in the two other families. Protein secretion tests showed that the R136W and I122T mutations inhibited secretion of the mutant proteins, whereas I359V had no effect on protein secretion; C179R was not tested, because of its predictable effect on protein folding. SIGNIFICANCE: These findings suggest that some LGI1 mutations may have a weak penetrance in families with complex inheritance pattern, or isolated patients, and that the protein secretion test, together with other predictive criteria, may help recognize pathogenic LGI1 mutations.

miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/ß-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/ß-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.

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 components form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signaling, 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 unexpected, 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 phenotype. 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 balance 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 supernumerary DRG sensory neurons in a wild-type background, and can rescue the sensory neuron 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's 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 lateral 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.

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the ß-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

ADAM23, a Gene Related to LGI1, Is Not Linked to Autosomal Dominant Lateral Temporal Epilepsy.

Autosomal dominant lateral temporal epilepsy (ADTLE) is an inherited epileptic syndrome characterized by ictal auditory symptoms or aphasia, negative MRI findings, and relatively benign evolution. Mutations responsible for ADLTE have been found in the LGI1 gene. The functions of the Lgi1 protein apparently are mediated by interactions with members of the ADAM protein family: it binds the postsynaptic receptor ADAM22 to regulate glutamate-AMPA currents at excitatory synapses and also the ADAM23 receptor to promote neurite outgrowth in vitro and dendritic arborization in vivo. Because alteration of each of these neuronal mechanisms may underlie ADLTE, ADAM22 and ADAM23 are candidate genes for this syndrome. In a previous work, we excluded a major role of ADAM22 in the aetiology of ADLTE. Here, we performed linkage analysis between microsatellite markers within or flanking the ADAM23 gene and ADLTE in 13 Italian families. The results exclude ADAM23 as major causative gene for ADLTE.

Association of intronic variants of the KCNAB1 gene with lateral temporal epilepsy.

The KCNAB1 gene is a candidate susceptibility factor for lateral temporal epilepsy (LTE) because of its functional interaction with LGI1, the gene responsible for the autosomal dominant form of LTE. We investigated association between polymorphic variants across the KCNAB1 gene and LTE. The allele and genotype frequencies of 14 KCNAB1 intronic SNPs were determined in 142 Italian LTE patients and 104 healthy controls and statistically evaluated. Single SNP analysis revealed one SNP (rs992353) located near the 3'end of KCNAB1 slightly associated with LTE after multiple testing correction (odds ratio=2.25; 95% confidence interval 1.26-4.04; P=0.0058). Haplotype analysis revealed two haplotypes with frequencies higher in cases than in controls, and these differences were statistically significant after permutation tests (Psim=0.047 and 0.034). One of these haplotypes was shown to confer a high risk for the syndrome (odds ratio=12.24; 95% confidence interval 1.32-113.05) by logistic regression analysis. These results support KCNAB1 as a susceptibility gene for LTE, in agreement with previous studies showing that this gene may alter susceptibility to focal epilepsy.