The identification of the Prox gene family and the development of optical projection tomography for use in Zebrafish

Vertebrate homologues of the Drosophila melanogaster gene prospero have been identified in many species. Whilst the function and regulation of prospero has been well studied in Drosophila the function and regulation of the homologous vertebrate gene, praxl, is not known. We describe the identification of the prox genes as members of a multigene family in vertebrates through the isolation of new members of the Prox gene family in zebrafish, Fugu rubripes, Tetraodon nigroviridis, mouse, and human. We examined the phylogeny of this new multigene family and we characterised the expression of these novel genes in zebrafish. Analysis of the expression of these genes identified the slow muscle as site of expression for prox 1 that did not overlap with the novel zebrafish Prox genes. Therefore, we studied the function ofprox 1 in the slow muscle using a combination of DNA, and morpholino injections. We demonstrate that prox 1 in not required for the specification of slow muscle as determined by the expression of markers of terminal differentiation. We also show that the medial lateral migration of the slow muscle is unaffected by the loss of prox 1. However, ectopic expression of prox 1 specifically in the fast muscle causes a defect in nuclear patterning. In normal development the fast muscle cells fuse early to form a multinucleate syncytium. The nuclei in this syncytium are normally evenly spaced. Ectopic expression of prox 1 resulted in the nuclei of the fast cells being positioned at the centre of the syncytium similarly to the situation observed in the mononucleate slow muscle. Furthermore loss of Prox 1 results in the disrupted patterning of the slow fibres, demonstrating a role for Prox 1 in the patterning of the slow muscle fibres. An understanding of the 3-dimensional (3D) pattern of gene expression can often lead to a better understanding of gene function. Optical projection tomography (OPT) is a new method for obtaining 3D data about an object. OPT generates a 3D digital model of a sample and allows it to be virtually sectioned, or rendered to produce a 3D image. OPT was developed for use on mouse embryos and had not been tested with zebrafish. We describe the difficulties of using OPT on samples as small as zebrafish embryos and the development of techniques to overcome these problems and allow its use in zebrafish

Established and emerging GABAA receptor pharmacotherapy for epilepsy

Drugs that modulate the GABAA receptor are widely used in clinical practice for both the long-term management of epilepsy and emergency seizure control. In addition to older medications that have well-defined roles for the treatment of epilepsy, recent discoveries into the structure and function of the GABAA receptor have led to the development of newer compounds designed to maximise therapeutic benefit whilst minimising adverse effects, and whose position within the epilepsy pharmacologic armamentarium is still emerging. Drugs that modulate the GABAA receptor will remain a cornerstone of epilepsy management for the foreseeable future and, in this article, we provide an overview of the mechanisms and clinical efficacy of both established and emerging pharmacotherapies

Commercially available industry-relevant software in the education of genome variant curation

BACKGROUND Genome analytics is a drastically expanding field, and there is an increasing demand for individuals with the necessary skillset to analyse the genome data that is being generated. A new Masters by coursework was developed to train individuals in genome analytics. Institutions analysing genomes commonly utilise in-house analysis tools, but increasingly commercial software packages that integrate AI are being considered in the research and diagnostic space (De La Vega et al., 2021). AIMS Evaluate the effectiveness of commercially available software as a tool for teaching variant analysis, classification and curation, enabling the analysis of real-world case examples for the teaching, training and assessment of students in the field of diagnostic genome analysis.   Assess students’ perspectives on work readiness after using commercially available tools in the educational environment. DESIGN AND METHODS Students were exposed to the use and limitations of a commercial software package for Human genome curation during two core units of the course. This software was used as part of both in-class training and in their assessment case studies. Students were invited to voluntarily complete an online survey including qualitative and quantitative components featuring Likert scale questions, both pre and post exposure to the software. Paired data from 23 individuals (73% response rate), most aged between 18 and 25, were recorded and anonymised prior to analysis. Qualitative data were thematically coded blind by two individuals independently using emergent coding (Charmaz, 2008). RESULTS AND CONCLUSIONS This project indicates that after the completion of the units that integrated commercially available industry software, we measured increased student confidence (increase in percentage reporting fairly confident or higher) in joining the genetic analysis workforce (significant change from 37% to 70%) and in completing job-specific tasks (significant increase in 7 out of 9 tasks of between 28% to 39%). The aspects of their studies the students valued in relation to these changes and their perception of the usefulness of integration of the commercial software were elucidated from qualitative theming, and can inform others looking to integrate commercially available software within their tertiary degree. REFERENCES Charmaz, K. (2008).  Grounded theory as an emergent method. In S. N. Hesse-Biber & P. Leavy (Eds.), Handbook of emergent methods. (pp. 155-170). The Guilford Press.  De La Vega, F.M., Chowdhury, S., Moore, B., Frise, E., McCarthy, J., Hernandez, E.J., Wong, T., James, K., Guidugli, L., Agrawal, P.B., Genetti, C.A., Brownstein, C.A., Beggs, A.H., Löscher, B.S., Franke, A., Boone, B., Levy, S.E., Õunap, K., Pajusalu, S., … Kingsmore, S.F. (2021). Artificial intelligence enables comprehensive genome interpretation and nomination of candidate diagnoses for rare genetic diseases. Genome Med, 13(1), 153. https://doi.org/10.1186/s13073-021-00965-

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases

Cadherin-Mediated Differential Cell Adhesion Controls Slow Muscle Cell Migration in the Developing Zebrafish Myotome

AbstractSlow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. Here we examine the cellular and molecular basis for slow-twitch muscle cell migration. We show that slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, which correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development

FishNet: an online database of zebrafish anatomy

Background: Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish. Results: To overcome this deficit we have utilized the technique of optical projection tomography to produce three-dimensional (3D) models of larval fish. In order to view and display these models we have created FishNet http://www.fishnet.org.au, an interactive reference of zebrafish anatomy spanning the range of zebrafish development from 24 h until adulthood. Conclusion: FishNet contains more than 36 000 images of larval zebrafish, with more than 1 500 of these being annotated. The 3D models can be manipulated on screen or virtually sectioned. This resource represents the first complete embryo to adult atlas for any species in 3D

Analysis of protein sequence and interaction data for candidate disease gene prediction

Linkage analysis is a successful procedure to associate diseases with specific genomic regions. These regions are often large, containing hundreds of genes, which make experimental methods employed to identify the disease gene arduous and expensive. We present two methods to prioritize candidates for further experimental study: Common Pathway Scanning (CPS) and Common Module Profiling (CMP). CPS is based on the assumption that common phenotypes are associated with dysfunction in proteins that participate in the same complex or pathway. CPS applies network data derived from protein–protein interaction (PPI) and pathway databases to identify relationships between genes. CMP identifies likely candidates using a domain-dependent sequence similarity approach, based on the hypothesis that disruption of genes of similar function will lead to the same phenotype. Both algorithms use two forms of input data: known disease genes or multiple disease loci. When using known disease genes as input, our combined methods have a sensitivity of 0.52 and a specificity of 0.97 and reduce the candidate list by 13-fold. Using multiple loci, our methods successfully identify disease genes for all benchmark diseases with a sensitivity of 0.84 and a specificity of 0.63. Our combined approach prioritizes good candidates and will accelerate the disease gene discovery process

Zebrafish prox1b Mutants Develop a Lymphatic Vasculature, and prox1b Does Not Specifically Mark Lymphatic Endothelial Cells

Background: The expression of the Prospero homeodomain transcription factor (Prox1) in a subset of cardinal venous cells specifies the lymphatic lineage in mice. Prox1 is also indispensible for the maintenance of lymphatic cell fate, and is therefore considered a master control gene for lymphangiogenesis in mammals. In zebrafish, there are two prox1 paralogues, the previously described prox1 (also known as prox1a) and the newly identified prox1b. Principal Findings: To investigate the role of the prox1b gene in zebrafish lymphangiogenesis, we knocked-down prox1b and found that depletion of prox1b mRNA did not cause lymphatic defects. We also generated two different prox1b mutant alleles, and maternal-zygotic homozygous mutant embryos were viable and did not show any lymphatic defects. Furthermore, the expression of prox1b was not restricted to lymphatic vessels during zebrafish development. Conclusion: We conclude that Prox1b activity is not essential for embryonic lymphatic development in zebrafish

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation