Neurobeachin Is Required Postsynaptically for Electrical and Chemical Synapse Formation

SummaryBackgroundNeural networks and their function are defined by synapses, which are adhesions specialized for intercellular communication that can be either chemical or electrical. At chemical synapses, transmission between neurons is mediated by neurotransmitters, whereas at electrical synapses, direct ionic and metabolic coupling occur via gap junctions between neurons. The molecular pathways required for electrical synaptogenesis are not well understood, and whether they share mechanisms of formation with chemical synapses is not clear.ResultsHere, using a forward genetic screen in zebrafish, we find that the autism-associated gene neurobeachin (nbea), which encodes a BEACH-domain-containing protein implicated in endomembrane trafficking, is required for both electrical and chemical synapse formation. Additionally, we find that nbea is dispensable for axonal formation and early dendritic outgrowth but is required to maintain dendritic complexity. These synaptic and morphological defects correlate with deficiencies in behavioral performance. Using chimeric animals in which individually identifiable neurons are either mutant or wild-type, we find that Nbea is necessary and sufficient autonomously in the postsynaptic neuron for both synapse formation and dendritic arborization.ConclusionsOur data identify a surprising link between electrical and chemical synapse formation and show that Nbea acts as a critical regulator in the postsynaptic neuron for the coordination of dendritic morphology with synaptogenesis

Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis

AbstractBackgroundTargeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.ResultsHere we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified.ConclusionsUsing this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.</p

Loss of Sbds in zebrafish leads to neutropenia and pancreas and liver atrophy

Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and skeletal abnormalities. Biallelic mutations in SBDS, which encodes a ribosome maturation factor, are found in 90% of SDS cases. Sbds–/– mice are embryonic lethal. Using CRISPR/Cas9 editing, we created sbds-deficient zebrafish strains. Sbds protein levels progressively decreased and became undetectable at 10 days postfertilization (dpf). Polysome analysis revealed decreased 80S ribosomes. Homozygous mutant fish developed normally until 15 dpf. Mutant fish subsequently had stunted growth and showed signs of atrophy in pancreas, liver, and intestine. In addition, neutropenia occurred by 5 dpf. Upregulation of tp53 mRNA did not occur until 10 dpf, and inhibition of proliferation correlated with death by 21 dpf. Transcriptome analysis showed tp53 activation through upregulation of genes involved in cell cycle arrest, cdkn1a and ccng1, and apoptosis, puma and mdm2. However, elimination of Tp53 function did not prevent lethality. Because of growth retardation and atrophy of intestinal epithelia, we studied the effects of starvation on WT fish. Starved WT fish showed intestinal atrophy, zymogen granule loss, and tp53 upregulation — similar to the mutant phenotype. In addition, there was reduction in neutral lipid storage and ribosomal protein amount, similar to the mutant phenotype. Thus, loss of Sbds in zebrafish phenocopies much of the human disease and is associated with growth arrest and tissue atrophy, particularly of the gastrointestinal system, at the larval stage. A variety of stress responses, some associated with Tp53, contribute to pathophysiology of SDS

Cilia-Associated Genes Play Differing Roles in Aminoglycoside-Induced Hair Cell Death in Zebrafish

Hair cells possess a single primary cilium, called the kinocilium, early in development. While the kinocilium is lost in auditory hair cells of most species it is maintained in vestibular hair cells. It has generally been believed that the primary role of the kinocilium and cilia-associated genes in hair cells is in the establishment of the polarity of actin-based stereocilia, the hair cell mechanotransduction apparatus. Through genetic screening and testing of candidate genes in zebrafish (Danio rerio) we have found that mutations in multiple cilia genes implicated in intraflagellar transport (dync2h1, wdr35, ift88, and traf3ip), and the ciliary transition zone (cc2d2a, mks1, and cep290) lead to resistance to aminoglycoside-induced hair cell death. These genes appear to have differing roles in hair cells, as mutations in intraflagellar transport genes, but not transition zone genes, lead to defects in kinocilia formation and processes dependent upon hair cell mechanotransduction activity. These mutants highlight a novel role of cilia-associated genes in hair cells, and provide powerful tools for further study

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA