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![F0 free-feeding wild-type and transgenic [Xtr.Tg (tubb2b:GFP) Amaya, RRID: EXRC_3001] Xenopus tropicalis crispants phenocopy key clinical hallmarks. Images show tadpole head structural morphology under normal conditions (a–c) and across the range of phenotypes observed: mild microcephaly (d–f), microcephaly with cataract(s) (g–i) and microcephaly with absent or missing eye structure(s) (j–l). White arrows on GFP fluorescence images show normal forebrain structures in un-injected control animals (b) and altered forebrain structures in mutants, with an increasing severity of microcephaly (e, h, k). Red arrows demonstrate the same forebrain structural trends in higher resolution MicroCT imaging (1% phosphotungstic acid contrast stain: c, f, i, l). Cataract formation is indicated in bright-field and MicroCT images by yellow arrows (g, i) and can be seen as a loss of GFP expression in fluorescence imaging (h), whilst green arrows highlight absent or missing structures of the eye (j, l). Re-occurring eye abnormalities were documented in 30 un-injected and 30 crispant tadpoles (exon 8, sgRNA3) to show the prevalence of cataract formation (mean, 14 tadpoles) and missing eye structures (mean, 5 tadpoles (m)). Further, 8 transgenic X. tropicalis crispant tadpole brains were imaged and measured (mm) 3 days post-fertilisation (control mean 1 mm, SD 0.02; Crispant mean 0.845 mm, SD 0.12 (t = 3.701; p = 0.007)) and 5 days post-fertilisation (control mean 6.37 mm, SD 0.29; Crispant mean 5.34 mm, SD 0.20 (t = 8.317; p = 0.000)) revealing sustained, significant reduction in brain length. Brain length measured as the distance from the forebrain to the hindbrain (Additional file 1: Fig. S5a) was expressed as a percentage of the mean of the control (N). Kaplan-Meier survival analysis of 50 un-injected control (median survival time 4.4 days) and crispant X. tropicalis tadpoles, injected at the one-cell stage with either sgRNA1 (exon 3 (Additional file 1: Fig. S4, median survival time 2 days) or sgRNA3 (exon 8, median survival time 3 days) show a significant difference in survival by log-rank comparison p = 0.000 (o)](publication/349615634/figure/fig5/AS:11431281115682457@1675051800305/F0-free-feeding-wild-type-and-transgenic-XtrTg-tubb2bGFP-Amaya-RRID-EXRC-3001.png)
F0 free-feeding wild-type and transgenic [Xtr.Tg (tubb2b:GFP) Amaya, RRID: EXRC_3001] Xenopus tropicalis crispants phenocopy key clinical hallmarks. Images show tadpole head structural morphology under normal conditions (a–c) and across the range of phenotypes observed: mild microcephaly (d–f), microcephaly with cataract(s) (g–i) and microcephaly with absent or missing eye structure(s) (j–l). White arrows on GFP fluorescence images show normal forebrain structures in un-injected control animals (b) and altered forebrain structures in mutants, with an increasing severity of microcephaly (e, h, k). Red arrows demonstrate the same forebrain structural trends in higher resolution MicroCT imaging (1% phosphotungstic acid contrast stain: c, f, i, l). Cataract formation is indicated in bright-field and MicroCT images by yellow arrows (g, i) and can be seen as a loss of GFP expression in fluorescence imaging (h), whilst green arrows highlight absent or missing structures of the eye (j, l). Re-occurring eye abnormalities were documented in 30 un-injected and 30 crispant tadpoles (exon 8, sgRNA3) to show the prevalence of cataract formation (mean, 14 tadpoles) and missing eye structures (mean, 5 tadpoles (m)). Further, 8 transgenic X. tropicalis crispant tadpole brains were imaged and measured (mm) 3 days post-fertilisation (control mean 1 mm, SD 0.02; Crispant mean 0.845 mm, SD 0.12 (t = 3.701; p = 0.007)) and 5 days post-fertilisation (control mean 6.37 mm, SD 0.29; Crispant mean 5.34 mm, SD 0.20 (t = 8.317; p = 0.000)) revealing sustained, significant reduction in brain length. Brain length measured as the distance from the forebrain to the hindbrain (Additional file 1: Fig. S5a) was expressed as a percentage of the mean of the control (N). Kaplan-Meier survival analysis of 50 un-injected control (median survival time 4.4 days) and crispant X. tropicalis tadpoles, injected at the one-cell stage with either sgRNA1 (exon 3 (Additional file 1: Fig. S4, median survival time 2 days) or sgRNA3 (exon 8, median survival time 3 days) show a significant difference in survival by log-rank comparison p = 0.000 (o)
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Background
Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”.
Methods
Whole exome or genome sequencing of two families with a...
Citations
... In recent years, CRISPR/Cas9 knockouts (KO) have been increasingly used to study gene function. In particular, CRISPR/Cas9 technology is available for the screening of candidate disease genes and creation of patient-specific mutations for human disease in Xenopus [27,156,157]. An advantage of working with X. laevis is the large body of literature on the genotype/phenotype correlations and the availability of injection and tested protocols optimized by the large community of researchers using X. laevis frogs as a model system in development and many other fields of research. ...
In vitro systems have been mainly promoted by authorities to sustain research by following the 3Rs principle, but continuously increasing amounts of evidence point out that in vivo experimentation is also of extreme relevance. Xenopus laevis, an anuran amphibian, is a significant model organism in the study of evolutionary developmental biology, toxicology, ethology, neurobiology, endocrinology, immunology and tumor biology; thanks to the recent development of genome editing, it has also acquired a relevant position in the field of genetics. For these reasons, X. laevis appears to be a powerful and alternative model to the zebrafish for environmental and biomedical studies. Its life cycle, as well as the possibility to obtain gametes from adults during the whole year and embryos by in vitro fertilization, allows experimental studies of several biological endpoints, such as gametogenesis, embryogenesis, larval growth, metamorphosis and, of course, the young and adult stages. Moreover, with respect to alternative invertebrate and even vertebrate animal models, the X. laevis genome displays a higher degree of similarity with that of mammals. Here, we have reviewed the main available literature on the use of X. laevis in the biosciences and, inspired by Feymann’s revised view, “Plenty of room for biology at the bottom”, suggest that X. laevis is a very useful model for all possible studies.
... The knockout of the Xenopus ortholog too, showed defects in the gonads and craniofacial region, phenocopying the human clinical defects (Brennand & Talkowski, 2021). The Xenopus is also a suitable model to study developmental events like neural development and left-right patterning (Duncan & Khokha, 2016) For example, model for holoprosencephaly and microcephaly have been developed using Xenopus, which reveal the underlying mechanisms of this NDD characterized by reduced number of NPCs and its renewal, increased apoptosis and overall impaired neurogenesis (Hoffmeister et al., 2017;Macken et al., 2021; Figure 3a). The true diploid, Xenopus tropicalis is ideal for loss of function studies whereas the tetraploid Xenopus laevis is ideal for gain of function and biochemical studies (Duncan & Khokha, 2016;Willsey et al., 2020). ...
The genesis and functioning of the central nervous system are one of the most intricate and intriguing aspects of embryogenesis. The big lacuna in the field of human CNS development is the lack of accessibility of the human brain for direct observation during embryonic and fetal development. Thus, it is imperative to establish alternative animal models to gain deep mechanistic insights into neurodevelopment, establishment of neural circuitry, and its function. Neurodevelopmental events such as neural specification, differentiation, and generation of neuronal and non‐neuronal cell types have been comprehensively studied using a variety of animal models and in vitro model systems derived from human cells. The experimentations on animal models have revealed novel, mechanistic insights into neurogenesis, formation of neural networks, and function. The models, thus serve as indispensable tools to understand the molecular basis of neurodevelopmental disorders (NDDs) arising from aberrations during embryonic development. Here, we review the spectrum of in vivo models such as fruitfly, zebrafish, frog, mice, and nonhuman primates to study neurogenesis and NDDs like microcephaly and Autism Spectrum Disorder. We also discuss nonconventional models such as ascidians and the recent technological advances in the field to study neurogenesis, disease mechanisms, and pathophysiology of human NDDs. This article is categorized under: Cancer > Stem Cells and Development Congenital Diseases > Stem Cells and Development Neurological Diseases > Stem Cells and Development Congenital Diseases > Genetics/Genomics/Epigenetics A spectrum of in vivo model organisms used to study CNS development and neurodevelopmental disorders – microcephaly and autism spectrum disorder.
... We therefore sought to establish and test a computational model of bioelectric pattern encoding in which we explicitly simulate bioelectrical states and gene-regulatory networks to understand their interplay. We chose to model the nascent Xenopus laevis brain, because it is a complex organ commonly used to model disorders with biomedical relevance [81][82][83][84][85][86][87] , and because its normal morphogenesis has already been shown to include a bioelectric component 20,[88][89][90] . Briefly, beginning at embryonic stage 16, a dramatic contrast develops between the transmembrane voltage potential at the neural plate (hyperpolarized) and in the surrounding ectoderm (depolarized), establishing a bioelectric pre-pattern that regulates subsequent large-scale brain development 88,89 (Fig. 1A-B). ...
Spatiotemporal bioelectric states regulate multiple aspects of embryogenesis. A key open question concerns how specific multicellular voltage potential distributions differentially activate distinct downstream genes required for organogenesis. To understand the information processing mechanisms underlying the relationship between spatial bioelectric patterns, genetics, and morphology, we focused on a specific spatiotemporal bioelectric pattern in the Xenopus ectoderm that regulates embryonic brain patterning. We used machine learning to design a minimal but scalable bioelectric-genetic dynamical network model of embryonic brain morphogenesis that qualitatively recapitulated previous experimental observations. A causal integration analysis of the model revealed a simple higher-order spatiotemporal information integration mechanism relating the spatial bioelectric and gene expression patterns. Specific aspects of this mechanism include causal apportioning (certain cell positions are more important for collective decision making), informational asymmetry (depolarized cells are more influential than hyperpolarized cells), long distance influence (genes in a cell are variably sensitive to voltage of faraway cells), and division of labor (different genes are sensitive to different aspects of voltage pattern). The asymmetric information-processing character of the mechanism led the model to predict an unexpected degree of plasticity and robustness in the bioelectric prepattern that regulates normal embryonic brain development. Our in vivo experiments verified these predictions via molecular manipulations in Xenopus embryos. This work shows the power of using a minimal in silico approach to drastically reduce the parameter space in vivo, making hard biological questions tractable. These results provide insight into the collective decision-making process of cells in interpreting bioelectric pattens that guide large-scale morphogenesis, suggesting novel applications for biomedical interventions and new tools for synthetic bioengineering.
... Through the prism of ARF-regulated mechanisms, the control of membrane transport by the GC thus appears critical for both development and homeostasis of differentiated tissues. Of interest, the particular sensibility of the nervous and/or skeletal systems to transport vesicles can also be seen in patients with genetic variants in the ARFGEFs BIG1 and BIG2 [15][16][17] , which promote ARF activation at the TGN, as well as in components of protein coats recruited by ARF proteins at both sides of the GC, such as COP-I coat proteins [18][19][20][21] or cargo adaptors of the AP complex family involved in cargo sorting at the TGN [22][23][24][25][26][27] (Table 1; Fig. 1). Again, the occurrence of both neonatal and progressive manifestations during childhood demonstrates the importance of the ARF pathway throughout development. ...
Association genetic studies and genome-scale CRISPR screens have recently identified ARF3 and TMEM251/LYSET/GCAF as Golgi-resident factors essential to brain and skeletal development. Here we discuss how even though the consequences of mutations in these genes affect endosomal and lysosomal compartments, the problem originates in the Golgi complex and may involve either the identity of the carrier vesicles or that of cargo molecules.
... Our model unifies multiple events triggered by depletion of the COPI complex and provides a framework for understanding the cellular impact of COPI malfunction or depletion in COPI-dependent genetic diseases, such as COPA syndrome, an immune dysregulatory disease characterized by polyarticular arthritis and progressive interstitial lung disease with pulmonary hemorrhages (Patwardhan and Spencer, 2019;Deng et al., 2020;Lepelley et al., 2020;Perrin et al., 2020), COPAassociated Alzheimer's disease development, (Astroski et al., 2021) and COPB1-dependent neurodevelopmental abnormalities (Macken et al., 2021). ...
The coatomer protein complex 1 (COPI) is a multi-subunit complex that coats intracellular vesicles and is involved in intracellular protein trafficking. Recently we and others found that depletion of COPI complex subunits zeta (COPZ1) and delta (ARCN1) preferentially kills tumor cells relative to normal cells. Here we delineate the specific cellular effects and sequence of events of COPI complex depletion in tumor cells. We find that this depletion leads to the inhibition of mitochondrial oxidative phosphorylation and elevation of ROS production, followed by accumulation of lipid droplets and autophagy-associated proteins LC3-II and SQSTM1/p62, and finally, apoptosis of the tumor cells. Inactivation of ROS in COPI-depleted cells with the mitochondrial-specific quencher, mitoquinone mesylate, attenuated apoptosis and markedly decreased both the size and number of lipid droplets. COPI depletion caused ROS-dependent accumulation of LC3-II and SQSTM1 which colocalizes with lipid droplets. Lack of double-membrane autophagosomes and insensitivity to Atg5 deletion suggested an accumulation of a microlipophagy complex on the surface of lipid droplets induced by depletion of the COPI complex. Our findings suggest a sequence of cellular events triggered by COPI depletion, starting with inhibition of oxidative phosphorylation, followed by ROS activation and accumulation of lipid droplets, and apoptosis.
... Gene editing in Xenopus species is now so efficient that analysis is routinely performed in founder animals, enabling rapid testing to support the causality of genetic disruption across a range of genes. [46][47][48][49] To support the use of X. tropicalis to model variants in gria1, both human and Xenopus tropicalis share identical gene structures and produce proteins that are >87% conserved (Figures S1A and S1B). Additionally, the exon 8 target region corresponding to the homozygous nonsense variant identified in individual 1 (p.Arg377Ter) is well conserved in Xenopus ( Figure S1C). ...
GRIA1 encodes the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, which are ligand-gated ion channels that act as excitatory receptors for the neurotransmitter L-glutamate (Glu). AMPA receptors (AMPARs) are homo- or heteromeric protein complexes with four subunits, each encoded by different genes, GRIA1 to GRIA4. Although GluA1-containing AMPARs have a crucial role in brain function, the human phenotype associated with deleterious GRIA1 sequence variants has not been established. Subjects with de novo missense and nonsense GRIA1 variants were identified through international collaboration. Detailed phenotypic and genetic assessments of the subjects were carried out and the pathogenicity of the variants was evaluated in vitro to characterize changes in AMPAR function and expression. In addition, two Xenopus gria1 CRISPR-Cas9 F0 models were established to characterize the in vivo consequences. Seven unrelated individuals with rare GRIA1 variants were identified. One individual carried a homozygous nonsense variant (p.Arg377Ter), and six had heterozygous missense variations (p.Arg345Gln, p.Ala636Thr, p.Ile627Thr, and p.Gly745Asp), of which the p.Ala636Thr variant was recurrent in three individuals. The cohort revealed subjects to have a recurrent neurodevelopmental disorder mostly affecting cognition and speech. Functional evaluation of major GluA1-containing AMPAR subtypes carrying the GRIA1 variant mutations showed that three of the four missense variants profoundly perturb receptor function. The homozygous stop-gain variant completely destroys the expression of GluA1-containing AMPARs. The Xenopus gria1 models show transient motor deficits, an intermittent seizure phenotype, and a significant impairment to working memory in mutants. These data support a developmental disorder caused by both heterozygous and homozygous variants in GRIA1 affecting AMPAR function.
... Although the role of proteins in specific diseases has often been studied by production of gene knockout (KO) cells or animals, many patients present mutated protein variants rather than complete absence of gene product. The application of NGS technologies such as whole genome/exome sequencing (WGS/WES) has been (Ng et al., 2009;Ng et al., 2010), and continues to be (Macken et al., 2021;Marom et al., 2021;Usmani et al., 2021) instrumental in the identification of novel mutations including those leading to splicing defects. Further, this mutational information can be efficiently analyzed using advanced algorithms to predict non-tolerated/pathogenic changes (Ng and Henikoff, 2003;Schwarz et al., 2010;Adzhubei et al., 2013;Ioannidis et al., 2016;Rentzsch et al., 2019;Ge et al., 2021) and by molecular dynamics to infer putative structural alterations (Kellogg et al., 2011;Adolf-Bryfogle and Dunbrack, 2013). ...
... In X. tropicalis, Nakayama and colleagues laid the foundation and set out a simple CRISPR pipeline and use of mutations in the tyrosinase gene to generate albinism phenotypes, targeting the start codon, leading to frameshift mutation and KO (Nakayama et al., 2013). CRISPR can be used to analyse gene function, and to replicate human disease mutations to generate mosaic targeted mutant F0's and lines in Xenopus embryos (Feehan et al., 2019;Macken et al., 2021;Naert et al., 2017Naert et al., , 2020Naert and Vleminckx, 2018;Nakayama et al., 2013). ...
In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20–22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and −219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively ‘drop out’ a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. miRNA-219 and miR-196a KO’s both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.
... A recent study showed that damage caused by oxidative stress in the lens of the eye is a major cause of cataracts. 3 Oxidative stress caused by exposure of lens epithelial cells to hydrogen peroxide (H 2 O 2 ) can lead to DNA damage and impair cell and tissue function. 4 H 2 O 2 is a non-free radical member of the reactive oxygen family, which produces hydroxyl radicals that irreversibly damage the lens epithelium, leading to cell death and cataract formation. ...
Opacity of the lens caused by cataracts could lead to severe visual impairment and even blindness. Oxidative stress caused by exposure of lens epithelial cells to hydrogen peroxide (H 2 O 2 ) can lead to DNA damage and impair cell function. Therefore, how to prevent lens epithelial cells from being harmed by H 2 O 2 is an urgent problem. The ZNF219 gene belongs to the Kruppel like zinc finger gene family, which is involved in a variety of biological processes. In this study, we found the low expression of ZNF219 in H 2 O 2 -induced HLE-B3 cells. We further noticed ZNF219 could improve the survival rate of H 2 O 2 -induced HLE-B3 cells, and inhibit the apoptosis and oxidative stress response. Mechanically, ZNF219 protected human lens epithelial cells against H 2 O 2 -induced injury via targeting SOX9 through activating AKT/GSK3β pathway. We therefore thought ZNF219 was a key protective protein in the oxidative damage of human lens epithelial cells and the pathogenesis of cataract.
... The detected transport proteins also included endosomal trafficking-related proteins, such as DnaJ homolog subfamily C member 13 (DNAJC13) [16,17] (Fig. 2A), which is involved in membrane trafficking through early endosomes and implicated in recycling epidermal growth factor receptor. Coatomer subunit beta (COPB1) [18,19] (Fig. 2A) is a cytosolic protein that associates with vesicles from the Golgi apparatus and mediating protein transport from the endoplasmic reticulum. BM-MSC EV cargo contained proteins involved in electron transport that have been shown to be co-expressed together in independent [20], a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase, which plays a critical role in the electron transport chain, was co-expressed with cytochrome b (MT-CYB) [21] (Fig. 2B), which is a component of the ubiquinolcytochrome c reductase complex, also a critical component of the respiratory chain, ultimately contributing to the synthesis of ATP needed for cellular processes. ...
Background
Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown therapeutic potential in various in vitro and in vivo studies in cutaneous wound healing. Furthermore, there are ubiquitous studies highlighting the pro-regenerative effects of BM-MSC extracellular vesicles (BM-MSC EVs). The similarities and differences in BM-MSC EV cargo among potential healthy donors are not well understood. Variation in EV protein cargo is important to understand, as it may be useful in identifying potential therapeutic applications in clinical trials. We hypothesized that the donors would share both important similarities and differences in cargo relating to cell proliferation, angiogenesis, Wnt signaling, and basement membrane formation—processes shown to be critical for effective cutaneous wound healing.
Methods
We harvested BM-MSC EVs from four healthy human donors who underwent strict screening for whole bone marrow donation and further Good Manufacturing Practices-grade cell culture expansion for candidate usage in clinical trials. BM-MSC EV protein cargo was determined via mass spectrometry and Proteome Discoverer software. Corresponding proteomic networks were analyzed via the UniProt Consortium and STRING consortium databases.
Results
More than 3000 proteins were identified in each of the donors, sharing > 600 proteins among all donors. Despite inter-donor variation in protein identities, there were striking similarities in numbers of proteins per biological functional category. In terms of biologic function, the proteins were most associated with transport of ions and proteins, transcription, and the cell cycle, relating to cell proliferation. The donors shared essential cargo relating to angiogenesis, Wnt signaling, and basement membrane formation—essential processes in modulating cutaneous wound repair.
Conclusions
Healthy donors of BM-MSC EVs contain important similarities and differences among protein cargo that may play important roles in their pro-regenerative functions. Further studies are needed to correlate proteomic signatures to functional outcomes in cutaneous repair.
