in-vivo-whole-brain-imaging-zebrafish-larvae-using-three-dimensional

<em>In Vivo</em> Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the ...

Chungnam National University

Microdeletion syndromes are frequent causes of neuropsychiatric disorders leading to intellectual disability as well as autistic features accompanied by epilepsy and craniofacial anomalies. From comparative deletion mapping of the smallest microdeletion to date at 12q24.31, found in a patient with overlapping clinical features of 12q24.31 microdeletion syndrome, we narrowed the putative critical region to 445 kb containing seven genes, one microRNA, and one non-coding RNA. Zebrafish in situ hybridization and comprehensive transcript analysis of annotated genes in the panels of human organ and brain suggest that these are all candidates for neurological phenotypes excluding the gene HPD. This is also corroborated by synteny analysis revealing the conservation of the order of these six candidate genes between humans and zebrafish. Among them, we propose histone demethylase KDM2B and histone methyltransferase SETD1B as the two most plausible candidate genes involved in intellectual disability, autism, epilepsy, and craniofacial anomalies. These two chromatin modifiers located approximately 224 kb apart were both commonly deleted in six patients, while two additional patients had either KDM2B or SETD1B deleted. The four additional candidate genes (ORAI1, MORN3, TMEM120B, RHOF), a microRNA MIR548AQ, and a non-coding RNA LINC01089 are localized between KDM2B and SETD1B. The 12q24.31 microdeletion syndrome with syndromic intellectual disability extends the growing list of microdeletion syndromes and underscores the causative roles of chromatin modifiers in cognitive and craniofacial development.

An atypical 12q24.31 microdeletion implicates six genes including a histone demethylase KDM2B and a histone methyltransferase SETD1B in syndromic intellectual disability.

Leukodystrophy with vanishing white matter (VWM), also called Childhood Ataxia with Central Nervous System Hypomyelination, is caused by mutations in the subunits of the eukaryotic translation initiation factor, EIF2B1, EIF2B2, EIF2B3, EIF2B4 or EIF2B5. However, little is known regarding the underlying pathogenetic mechanisms, and there is no curative treatment for VWM. In this study, we established the first EIF2B3 animal model for VWM disease in vertebrates by CRISPR mutagenesis of the highly conserved zebrafish ortholog eif2b3. Using CRISPR, we generated two mutant alleles in zebrafish eif2b3, 10- and 16-bp deletions, respectively. The eif2b3 mutants showed defects in myelin development and glial cell differentiation, and increased expression of genes in the induced stress response pathway. Interestingly, we also found ectopic angiogenesis and increased VEGF expression. Ectopic angiogenesis in the eif2b3 mutants was reduced by the administration of VEGF receptor inhibitor SU5416. Using the eif2b3 mutant zebrafish model together with in silico protein modeling analysis, we demonstrated the pathogenicity of 18 reported mutations in EIF2B3, as well as of a novel variant identified in a 19-month-old female patient: c.503 T > C (p.Leu168Pro). In summary, our zebrafish mutant model of eif2b3 provides novel insights into VWM pathogenesis and offers rapid functional analysis of human EIF2B3 gene variants.

Eif2b3 mutants recapitulate phenotypes of vanishing white matter disease and validate novel disease alleles in zebrafish.

Three-dimensional fluorescence microscopy has an intrinsic performance limit set by the number of photons that can be collected from the sample in a given time interval. Here, we extend our earlier work - a recursive light propagation network (RLP-Net) - which is a computational microscopy technique that overcomes such limitations through virtual refocusing that enables volume reconstruction from two adjacent 2-D wide-field fluorescence images. RLP-Net employs a recursive inference scheme in which the network progressively predicts the subsequent planes along the axial direction. This recursive inference scheme reflects that the law of physics for the light propagation remains spatially invariant and therefore a fixed function (i.e., a neural network) for a short distance light propagation can be recursively applied for a longer distance light propagation. In addition, we employ a self-supervised denoising method to enable accurate virtual light propagation over a long distance. We demonstrate the capability of our method through high-speed volumetric imaging of neuronal activity of a live zebrafish brain. The source code used in the paper is available at https://github.com/NICALab/rlpnet.

Article	Year
An atypical 12q24.31 microdeletion implicates six genes including a histone demethylase KDM2B and a histone methyltransferase SETD1B in syndromic intellectual disability. Human genetics\| PubMed ID: 27106595	2016
Eif2b3 mutants recapitulate phenotypes of vanishing white matter disease and validate novel disease alleles in zebrafish. Human molecular genetics\| PubMed ID: 33517449	2021
Three-dimensional fluorescence microscopy through virtual refocusing using a recursive light propagation network. Medical image analysis\| PubMed ID: 36116298	2022

Kang-Han Lee

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