Knockout Zebrafish Research Articles

Deletion of c16orf45 in zebrafish results in a low fertilization rate and increased thigmotaxis.

c16orf45 is located at 16p13.11, an important locus related to neurodevelopmental diseases. Clinical studies have demonstrated that c16orf45 is associated with various neurodevelopmental diseases. To further elucidate the effect of c16orf45 on neural development, we constructed a zebrafish model with a stably inherited c16orf45 deletion via CRISPR/Cas9 technology. We found that deletion of c16orf45 significantly reduced the zebrafish fertilization rate, and both females and males showed reduced fertility. Meanwhile, the homozygous c16orf45 knockout zebrafish showed a developmental delay at 24hr postfertilization (hpf). However, morphological changes were not apparent after 2days postfertilization (dpf). Notably, the results of behavioral experiments revealed increased thigmotaxis in c16orf45-/- zebrafish at 2months. In conclusion, these findings demonstrate that c16orf45 plays an important role in nervous system and reproductive system.

Identification of KIAA0196 as a novel susceptibility gene for myofibril structural disorganization in cardiac development

BackgroundCongenital heart disease is one of the most common cardiac malformation-related diseases worldwide. Some causative genes have been identified but can explain only a small proportion of all cases; therefore, the discovery of novel susceptibility genes and/or modifier genes for abnormal cardiac development remains a major challenge. MethodsWe used a single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS) was conducted to screen and quickly identify candidate genes. KIAA0196 knockout zebrafish and mice were generated by CRISPR/Cas9 to detect whether or how KIAA0196 deficiency would influence cardiac development. ResultsHomozygous, but not heterozygous, zebrafish and mice showed early embryonic lethality. At the embryonic stage, microscopic examination and dissection revealed pericardial edema and ventricle enlargement in homozygous zebrafish and obviously delayed cardiac development in heterozygous mice, while echocardiography and tissue staining showed that significantly decreased cardiac function, ventricle enlargement, myofibril loss, and significantly reduced trabecular muscle density were observed in adult heterozygous zebrafish and mice. Most importantly, immunostaining and electron microscopy showed that there was a significant increase in sarcomere structural disorganization and myofibril structural integrity loss in KIAA0196 mutants. Furthermore, substantial downregulation in other sarcomeric genes and proteins was detected and verified in a mouse model via transcriptome and proteomics analyses; these changes especially affected the myosin heavy or light chain (MYH or MYL) family genes. ConclusionWe identified KIAA0196 for the first time as a susceptibility gene for abnormal cardiac development. KIAA0196 deficiency may cause abnormal heart development by influencing the structural integrity of myofibrils.

Role of GUCA1C in Primary Congenital Glaucoma and in the Retina: Functional Evaluation in Zebrafish.

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.

MON-714 CRISPR / Cas9 Genomic Edition of the CDH2 Gene in Zebrafish Leads to Eye and Cardiac Malformation

Introduction: The CDH2 gene encodes a transmembrane protein responsible for calcium-dependent cell-cell adhesion that participates in the process of embrionic development. Neural development is achieved by neuronal neurulation, migration and differentiation, as well as axon growth and synapse formation. During pituitary development, CDH2 was associated to the epithelium-to-mesenchymal transition, with cell migration from the marginal zone to the anterior pituitary gland, followed by terminal differentiation. Thus, a dysfunction in n-cadherins disrupts the architecture of the neural tube, cortical architecture of the embryonic brain and pituitary development. In a previous study in our laboratory, a patient was diagnosed with a homozygous variant located in the N-terminal region of CDH2 (p.Val289IIe) that culminated in congenital hypopituitarism and pituitary hypoplasia. Together, these observations indicate that cadherins, especially N-cadherin, play an indispensable role in the organization of neuroepithelial layers. Zebrafish has been widely used as a model for studies of gene functionality, as it has 70% genetic homology to humans, besides being a small animal with rapid development. Our goal was to generate a zebrafish knockout for the CDH2 gene, using CRISPR Cas9 genomic edition to study its importance during development and analyze this gene in patients with characteristics similar to those observed in zebrafish. Material and methods: Three guides were drawn for the CDH2 gene using crispor program. sgRNA, produced by in vitro transcription, and Cas9 protein were injected into one cell stage. All developmental parameters were observed under a microscope up to 96 hours post fertilization (hpf). Mortality rate were calculated at 24, 48, 72 and 96 hpf. The embryos were genotyped to confirm the deleterious allelic variant. CDH2 coding region was evaluated in 3 female siblings, born from consanguineous parents, presenting micro/anophtalmia and short stature. Exons 2 to 16 were sequenced by the Sanger method. Results: 352 eggs were injected and several deformities such as absence of somites, cardiac edema, spinal curvature, cranial malformation and microphthalmia or total absence of eyes were observed. The mortality rates were 26%, 31%, 40% and 62% at 24, 48, 72 and 96 hpf, respectively. The Sanger sequencing from DNA extracted from the whole animal presented deleterious effect classified as insertions, deletions and missense changes. No deleterious allelic variant was observed in the 15 analyzed exons in the 3 patients. Conclusion: The CDH2 gene is important for neurodevelopment and eyes formation in the zebrafish although pathogenic allelic variants in this gene was not found in the studied patients with short stature and eye abnormalities.

Heat-shock-induced tyrosinase gene ablation with CRISPR in zebrafish.

Tyrosinase (TYR) converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA into L-dopaquinone, which can produce melanin pigment. The abrogation of the functional activity of TYR can result in albino skin and eye diseases because of a deficiency in melanin pigment production. In this study, we developed and characterized an inducible knockout TYR platform comprising the heat-inducible heat-shock-promoter-70-driving CRISPR/Cas9 system and a zU6-promoter-driving tyr single guide RNA (sgRNA) system to investigate the temporal expression of TYR genes. To overcome the difficulty of identifying zebrafish germline integrations and facilitate the observation of Cas9 expression, heart-specific cmlc2:enhanced green fluorescent protein (EGFP; used to confirm tyr sgRNA expression) and two selectable markers (P2A-mCherry and internal ribosomal entry site-EGFP) were applied in our system. Heat shock treatment administered to Cas9 transgenic embryos induced mCherry or EGFP fluorescence expression throughout the embryos' bodies, and Cas9 protein was detected 1h after heat shock treatment. Mutations were created by direct injection and line crossing, which led to mosaic and complete depigmentation phenotypes in approximately 50% and 100% of the embryos, respectively. Using our system, conditional TYR knockout in zebrafish was achieved efficiently and simply.

A Novel Function of the Lysophosphatidic Acid Receptor 3 (LPAR3) Gene in Zebrafish on Modulating Anxiety, Circadian Rhythm Locomotor Activity, and Short-Term Memory

Lysophosphatidic acid (LPA) is a small lysophospholipid molecule that activates multiple cellular functions through pathways with G-protein-coupled receptors. So far, six LPA receptors (LPAR1 to LPAR6) have been discovered and each one of them can connect to the downstream cell message-transmitting network. A previous study demonstrated that LPA receptors found in blood-producing stem cells can enhance erythropoietic processes through the activation of LPAR3. In the current study, newly discovered functions of LPAR3 were identified through extensive behavioral tests in lpar3 knockout (KO) zebrafish. It was found that the adult lpar3 KO zebrafish display an abnormal movement orientation and altered exploratory behavior compared to that of the control group in the three-dimensional locomotor and novel tank tests, respectively. Furthermore, consistent with those results, in the circadian rhythm locomotor activity test, the lpar3 KO zebrafish showed a lower level of angular velocity and average speed during the light cycles, indicating an hyperactivity-like behavior. In addition, the mutant fish also exhibited considerably higher locomotor activity during the dark cycle. Supporting those findings, this phenomenon was also displayed in the lpar3 KO zebrafish larvae. Furthermore, several important behavior alterations were also observed in the adult lpar3 KO fish, including a lower degree of aggression, less interest in conspecific social interaction, and looser shoal formation. However, there was no significant difference regarding the predator avoidance behavior between the mutant and the control fish. In addition, lpar3 KO zebrafish displayed memory deficiency in the passive avoidance test. These in vivo results support for the first time that the lpar3 gene plays a novel role in modulating behaviors of anxiety, aggression, social interaction, circadian rhythm locomotor activity, and memory retention in zebrafish.

Zebrafish cyp17a1 knockout reveals that androgen-mediated signaling is important for male brain sex differentiation

Brain sex differentiation is a complex process, wherein genes and steroid hormones act to induce specific gender brain differentiation. Testosterone (T) derived from the gonads has been linked to neural circuit modeling in a sex-specific manner. Previously, we have shown that cyp17a1 knockout (KO) zebrafish have low plasma androgen levels, and display compromised male-typical mating behaviors. In this study, we demonstrated that treatment of cyp17a1 KO males with T or 11-ketotestosterone (11-KT) is sufficient to rescue mating impairment by restoring the male-typical secondary sex characters (SSCs) and mating behaviors, confirming an essential role of androgen in maintaining SSCs and mating behaviors. Brain steroid hormone analysis revealed that cyp17a1 KO fish have reduced levels of T and 11-KT. We performed RNA sequencing on brain samples of control and cyp17a1 KO male zebrafish to get insights regarding the impact of cyp17a1 KO on gene expression pattern, and to correlate it with the observed disruption of male-typical mating behaviors. Transcriptome analysis of cyp17a1 KO males showed a differential gene expression when compared to control males. In total, 358 genes were differentially regulated between control males and KO males. Important genes including brain aromatase (cyp19a1b), progesterone receptor (pgr), deiodinase (dio2), and insulin-like growth factor 1 (igf1) that are involved in brain functions, as well as androgen response genes including igf1, frem1a, elovl1a, pax3a, mmp13b, hsc70, ogg1 were regulated. RT-qPCR analysis following rescue of cyp17a1 KO with T and 11-KT further suggested that androgen-mediated signaling is disrupted in the cyp17a1 KO fish. Our results indicated that cyp17a1 KO fish have an incomplete masculinization and altered brain gene expression, which could be due to decreased androgen levels.

Mutations of RNF213 are responsible for sporadic cerebral cavernous malformation and lead to a mulberry-like cluster in zebrafish

Although familial forms of cerebral cavernous malformation are mainly attributed to three CCM genes (KRIT1, CCM2 and PDCD10), no mutation is identified in sporadic cerebral cavernous malformation cases with a unique lesion, indicating additional genes for sporadic cerebral cavernous malformation. To screen the candidate genes, we conducted whole exome sequencing in 31 sporadic cerebral cavernous malformation patients and 32 healthy controls, and identified 5 affected individuals carrying 6 heterozygous deleterious mutations in RNF213 but no RNF213 mutation in healthy individuals. To further confirm RNF213 was associated with cerebral cavernous malformation, we generated rnf213a homozygous knockout zebrafish and found mutation of rnf213a in zebrafish led to a mulberry-like cluster of disordered-flow vascular channels which was reminiscent of human cerebral cavernous malformation. In addition, we revealed kbtbd7 and anxa6 were significantly downregulated due to rnf213a mutation through transcriptomic sequencing and RT-qPCR analysis. Based on the mulberry-like phenotype partly rescued by mRNA of kbtbd7 as well as anxa6, we suggested that rnf213a promoted mulberry-like cluster via downregulation of kbtbd7 and anxa6. Altogether, we firstly demonstrate RNF213is a novel candidate gene for sporadic cerebral cavernous malformation and the mutation of rnf213a is responsible for the mulberry-like cluster in zebrafish.

Integration of GPCR‐induced endothelial cytoprotection signaling by β‐arrestin‐2

Vascular dysfunction is a hallmark of inflammation and results in endothelial barrier disruption, vascular leakage and apoptosis. G protein‐coupled receptors (GPCRs) are highly druggable and mediate inflammatory responses associated with endothelial dysfunction. Specifically, two GPCRs, protease‐activated receptor‐1 (PAR1) and sphingosine‐1‐phosphate receptor‐1 (S1PR1) promote endothelial cytoprotection including barrier protection, Akt‐mediated cell survival, and anti‐inflammatory responses. Activated protein C (APC) activates PAR1 to enhance endothelial barrier stability, whereas S1PR1 contributes to basal barrier stability, whether the two receptors coordinate signaling to promote endothelial cytoprotection is not known. Our previous work showed that APC/PAR1 signaling via the β‐arrestin‐2 and Dishevelled‐2 (Dvl2) scaffolds promote Rac1 signaling and endothelial barrier protection. However, whether β‐arrestin‐2 and Dvl2 are required for integration of S1PR1 into APC/PAR1‐mediated cytoprotection is not known. Here, we report that APC/PAR1 protection against TNF‐α‐induced apoptosis requires S1PR1, whereas S1PR1 is not necessary for APC/PAR1‐mediated barrier protection. We further show that APC/PAR1 transactivation of S1PR1 is mediated by β‐arrestin‐2 dependent activation of sphingosine kinase 1 (Sphk1) and is pivotal for anti‐apoptotic Akt signaling. S1PR1, is activated by sphingosine‐1‐phosphate generated by Sphk1 and we hypothesized that β‐arrestin‐2 and Dvl2 were required for Sphk1 activity. We found that APC promotes Sphk1 phosphorylation and translocation to the membrane. In addition, loss of β‐arrestin‐2 but not Dvl2 abolished APC‐mediated Sphk1 activation. Pharmacological antagonism of S1PR1, inhibition of Sphk1, and β‐arrestin‐2 depletion abolished APC/PAR1‐mediated Akt signaling. Moreover, inhibition of Sphk1, and β‐arrestin‐2 depletion also resulted in loss of APC/PAR1‐mediated cell survival suggesting that Sphk1 and β‐arrestin‐2 are involved in APC/PAR1‐mediated cytoprotection. Our mechanistic studies were corroborated in vivo using a Sphk1 gene knockout zebrafish model. Loss of Sphk1 ablated APC/PAR1‐mediated cell survival and we are currently in the process of validating a β‐arrestin‐2 knockout zebrafish line to test its role in vivo in cytoprotection. In summary, our work demonstrates that APC/PAR1 signaling via the β‐arrestin‐2 multi‐functional scaffold segregates into two distinct pathways: 1) β‐arrestin‐2‐Sphk1 activation induces transactivation of S1PR1 to promote Akt‐mediated cell survival and 2) β‐arrestin‐2/Dvl2‐Rac1 activation promotes endothelial barrier protection.Support or Funding InformationSupported by NIH/ NIGMS R35 GM127121 and NIGMS IRACDA K12 GM068524.

Examining the Role of the chromatin remodeler CHD5 in Tumor Suppression and Neural Differentiation in Zebrafish

Gene expression and neurogenesis in vertebrates involves a wide variety of cellular machinery to coordinate the transcriptional changes that direct cellular differentiation. Critical aspects of this epigenetic machinery are chromatin remodeling factors. One such factor is CHD5, a vertebrate‐specific member of a family of ATP‐dependent chromatin remodeling proteins. CHD5 is expressed primarily in neural tissue where it is thought to contribute to neurogenesis and has been strongly linked to tumor suppression. Currently, little is known regarding the molecular mechanisms by which CHD5 contributes to neurogenesis or tumor suppression. We hypothesize that loss of CHD5 contributes to development of neuroblastoma because neural precursor cells lacking CHD5 fail to alter some aspect of their neural precursor transcriptome/epigenome. To study the biochemical properties and functional importance of CHD5 remodelers, we have developed a zebrafish model to study Chd5, the zebrafish homolog of human CHD5.Using the CRISPR/Cas9 system, we have engineered chd5 knockout zebrafish embryos. These zebrafish did not display any overt developmental phenotypes, however next‐generation sequencing of brain tissue from zebrafish embryos reveals that loss of chd5 is associated with broad changes in gene expression. These genes are both up and down regulated, suggesting Chd5 is necessary for both activation and repression of genes during brain development in zebrafish. Analysis of these genes and of their possible contribution to Chd5‐dependent phenotypes is underway.Support or Funding InformationPurdue Center for Cancer Research; Purdue College of Agriculture

MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP–bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site subpocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1-catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.

Pannexin-1 mediates fluid shear stress-sensitive purinergic signaling and cyst growth in polycystic kidney disease.

Tubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/- ) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1-/- mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1-/- mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed. Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD.

Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes (rnf185 and rnf215) were successfully mutated in F1 fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear.

Knockout of DNase1l1l abrogates lens denucleation process and causes cataract in zebrafish

Removal of nuclei in lens fiber cells is required for organelle-free zone (OFZ) formation during lens development. Defect in degradation of nuclear DNA leads to cataract formation. DNase2β degrades nuclear DNA of lens fiber cells during lens differentiation in mouse. Hsf4 is the principal heat shock transcription factor in lens and facilitates the lens differentiation. Knockout of Hsf4 in mouse and zebrafish resulted in lens developmental defect that was characterized by retaining of nuclei in lens fiber cells. In previous in vitro studies, we found that Hsf4 promoted DNase2β expression in human and mouse lens epithelial cells. In this study, it was found that, instead of DNase2β, DNase1l1l is uniquely expressed in zebrafish lens and was absent in Hsf4−/− zebrafish lens. Using CRISPR-Cas9 technology, a DNase1l1l knockout zebrafish line was constructed, which developed cataract. Deletion of DNase1l1l totally abrogated lens primary and secondary fiber cell denucleation process, whereas had little effect on the clearance of other organelles. The transcriptional regulation of DNase1l1l was dramatically impaired in Hsf4−/− zebrafish lens. Rescue of DNase1l1l mRNA into Hsf4−/− zebrafish embryos alleviated its defect in lens fiber cell denucleation. Our results in vivo demonstrated that DNase1l1l is the primary DNase responsible for nuclear DNA degradation in lens fiber cells, and Hsf4 can transcriptionally activate DNase1l1l expression in zebrafish.

Glucocorticoid and mineralocorticoid receptor activation modulates postnatal growth.

During early development, stress or exogenous glucocorticoid (GC) administration reduces body mass in vertebrates, and this is associated with the glucocorticoid receptor (GR) activation. Although GCs also activate the mineralocorticoid receptor (MR), the physiological significance of MR activation on early developmental growth is unknown. We tested the hypothesis that activation of both GR and MR are required for postnatal growth suppression by GCs. Differential regulation of GR and MR activation was achieved by using ubiquitous GR- (GRKO) and MR- (MRKO) knockout zebrafish (Danio rerio) in combination with exogenous cortisol treatment. MR activation increased protein deposition in zebrafish larvae and also upregulated lepa and downregulated lepr transcript abundance. Cortisol treatment reduced body mass and protein content in the WT, and this corresponded with the upregulation of muscle proteolytic markers, including murf1 and redd1 by GR activation. The combined activation of MR and GR by cortisol also upregulated the gh and igf1 transcript abundance, and insulin expression compared to the WT. However, cortisol-mediated reduction in body mass and protein content required the activation of both MR and GR, as activation by GR alone (MRKO + cortisol) did not reduce the larval protein content. Collectively, our results indicate that MR activation favors protein deposition and GR activation stimulates proteolysis, while their combined activation is involved in cortisol-mediated growth suppression. Overall, this work provides insight into the physiological significance of MR activation in regulating protein deposition during early development at a systems level.

Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases.

The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as 'pathogenic' by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from 'Uncertain significance' to 'Likely pathogenic' in six probands. Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.

Dact2 is involved in the regulation of epithelial-mesenchymal transition

Dishevelled-associated antagonist of beta-catenin 2 (Dact2) is involved in the regulation of intracellular signaling pathways during development. It negatively regulates the Nodal signaling pathway, possibly by promoting lysosomal degradation of Nodal receptors such as TGFBR1, and plays an inhibitory role during the re-epithelialization of skin wounds by attenuating transforming growth factor-β signaling. Dact2 is known to act as a functional tumor suppressor in colon cancer; reduced Dact2 can promote liver cancer progression and suppress gastric cancer proliferation, invasion, and metastasis by inhibiting Wnt signaling. Zebrafish is used as a model of cancer biology because it shows similar tumorigenesis and morphogenesis as in humans and gene manipulation in this organism is possible. This study was performed to explore phenotypic changes in Dact2 knockout zebrafish and investigate the function of Dact2. A 10-base pair deletion Dact2 knockout zebrafish was prepared using the CRISPR-Cas9 genome editing system. Dact2 knockout enhanced the expression of the MMP2 and MMP9 genes, which are related to tumor invasion and migration, and the Snail, VEGF, and ZEB genes, which are related to epithelial-mesenchymal transition (EMT). The absence of Dact2 also resulted in hyperplasia of the gastrointestinal epithelium, fibrosis in the pancreas and liver, increased proliferation of the pancreatic and hepatic bile ducts, and invasive proliferation into the pancreas. A wound healing assay confirmed that the absence of Dact2 enhanced EMT, thus accelerating wound healing. This study suggests that a loss of function of Dact2 impacts EMT-related gene regulation and tumor generation in a zebrafish knockout model, which is a useful model for exploring the mechanisms of these processes.

The novel zebrafish model pretzel demonstrates a central role for SH3PXD2B in defective collagen remodelling and fibrosis in Frank-Ter Haar syndrome.

ABSTRACTFrank-Ter Haar syndrome (FTHS, MIM #249420) is a rare skeletal dysplasia within the defective collagen remodelling spectrum (DECORS), which is characterised by craniofacial abnormalities, skeletal malformations and fibrotic soft tissues changes including dermal fibrosis and joint contractures. FTHS is caused by homozygous or compound heterozygous loss-of-function mutation or deletion of SH3PXD2B (Src homology 3 and Phox homology domain-containing protein 2B; MIM #613293). SH3PXD2B encodes an adaptor protein with the same name, which is required for full functionality of podosomes, specialised membrane structures involved in extracellular matrix (ECM) remodelling. The pathogenesis of DECORS is still incompletely understood and, as a result, therapeutic options are limited. We previously generated an mmp14a/b knockout zebrafish and demonstrated that it primarily mimics the DECORS-related bone abnormalities. Here, we present a novel sh3pxd2b mutant zebrafish, pretzel, which primarily reflects the DECORS-related dermal fibrosis and contractures. In addition to relatively mild skeletal abnormalities, pretzel mutants develop dermal and musculoskeletal fibrosis, contraction of which seems to underlie grotesque deformations that include kyphoscoliosis, abdominal constriction and lateral folding. The discrepancy in phenotypes between mmp14a/b and sh3pxd2b mutants suggests that in fish, as opposed to humans, there are differences in spatiotemporal dependence of ECM remodelling on either sh3pxd2b or mmp14a/b. The pretzel model presented here can be used to further delineate the underlying mechanism of the fibrosis observed in DECORS, as well as screening and subsequent development of novel drugs targeting DECORS-related fibrosis.This paper has an associated First Person interview with the first author of the article.

Dpysl2 (CRMP2) is required for the migration of facial branchiomotor neurons in the developing zebrafish embryo.

Dihydropyrimidinase-like family proteins (Dpysls) are relevant in several processes during nervous system development; among others, they are involved in axonal growth and cell migration. Dpysl2 (CRMP2) is the most studied member of this family; however, its role in vivo is still being investigated. Our previous studies in zebrafish showed the requirement of Dpysl2 for the proper positioning of caudal primary motor neurons and Rohon-Beard neurons in the spinal cord.In the present study, we show that Dpysl2 is necessary for the proper migration of facial branchiomotor neurons during early development in zebrafish. We generated a dpysl2 knock-out (KO) zebrafish mutant line and used different types of antisense morpholino oligonucleotides (AMO) to analyze the role of Dpysl2 in this process. Both dpysl2 KO mutants and morphants exhibited abnormalities in the migration of these neurons from rhombomers (r) 4 and 5 to 6 and 7. The facial branchiomotor neurons that were expected to be at r6 were still located at r4 and r5 hours after the migration process should have been completed. In addition, mutant phenotypes were rescued by injecting dpysl2 mRNA into the KO embryos. These results indicate that Dpysl2 is involved in the proper migration of facial branchiomotor neurons in developing zebrafish embryos.

Fgfr3 mutation disrupts chondrogenesis and bone ossification in zebrafish model mimicking CATSHL syndrome partially via enhanced Wnt/β-catenin signaling.

CATSHL syndrome, characterized by camptodactyly, tall stature and hearing loss, is caused by loss-of-function mutations of fibroblast growth factor receptors 3 (FGFR3) gene. Most manifestations of patients with CATSHL syndrome start to develop in the embryonic stage, such as skeletal overgrowth, craniofacial abnormalities, however, the pathogenesis of these phenotypes especially the early maldevelopment remains incompletely understood. Furthermore, there are no effective therapeutic targets for this skeleton dysplasia.Methods: We generated fgfr3 knockout zebrafish by CRISPR/Cas9 technology to study the developmental mechanisms and therapeutic targets of CATSHL syndrome. Several zebrafish transgenic lines labeling osteoblasts and chondrocytes, and live Alizarin red staining were used to analyze the dynamical skeleton development in fgfr3 mutants. Western blotting, whole mount in situ hybridization, Edu labeling based cell proliferation assay and Wnt/β-catenin signaling antagonist were used to explore the potential mechanisms and therapeutic targets.Results: We found that fgfr3 mutant zebrafish, staring from early development stage, showed craniofacial bone malformation with microcephaly and delayed closure of cranial sutures, chondroma-like lesion and abnormal development of auditory sensory organs, partially resembling the clinical manifestations of patients with CATSHL syndrome. Further studies showed that fgfr3 regulates the patterning and shaping of pharyngeal arches and the timely ossification of craniofacial skeleton. The abnormal development of pharyngeal arch cartilage is related to the augmented hypertrophy and disordered arrangement of chondrocytes, while decreased proliferation, differentiation and mineralization of osteoblasts may be involved in the delayed maturation of skull bones. Furthermore, we revealed that deficiency of fgfr3 leads to enhanced IHH signaling and up-regulated canonical Wnt/β-catenin signaling, and pharmacological inhibition of Wnt/β-catenin could partially alleviate the phenotypes of fgfr3 mutants.Conclusions: Our study further reveals some novel phenotypes and underlying developmental mechanism of CATSHL syndrome, which deepens our understanding of the pathogenesis of CATSHL and the role of fgfr3 in skeleton development. Our findings provide evidence that modulation of Wnt/β-catenin activity could be a potential therapy for CATSHL syndrome and related skeleton diseases.