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Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule‐induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using azido‐caged nucleobases that are site‐specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, efficiently blocks Watson–Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule‐induced gene knockdown in mammalian cells, zebrafish embryos, and frog embryos. We validated the general applicability of this approach by targeting three different genes. [ABSTRACT FROM AUTHOR]

Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two asb5 gene isoforms, asb5a and asb5b. Currently, the effects of asb5 gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated asb5a-knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar asb5a and asb5b expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target asb5b in wild-type and asb5a-knockout zebrafish. Knocking down asb5b in the wild-type had no phenotypic impact, but simultaneous asb5b knockdown in asb5a-knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in asb5a and asb5b, with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into asb5's mechanism. [ABSTRACT FROM AUTHOR]

Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved in vitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as in vivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional in vivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.

Oxytocin increases the salience of both positive and negative social contexts and it is thought that these diverse actions on behavior are mediated in part through circuit-specific action. This hypothesis is based primarily on manipulations of oxytocin receptor function, leaving open the question of whether different populations of oxytocin neurons mediate different effects on behavior. Here we inhibited oxytocin synthesis in a stress-sensitive population of oxytocin neurons specifically within the medioventral bed nucleus of the stria terminalis (BNSTmv). Oxytocin knockdown prevented social stress-induced increases in social vigilance and decreases in social approach. Viral tracing of BNSTmv oxytocin neurons revealed fibers in regions controlling defensive behaviors, including lateral hypothalamus, anterior hypothalamus, and anteromedial BNST (BNSTam). Oxytocin infusion into BNSTam in stress naïve mice increased social vigilance and reduced social approach. These results show that a population of extrahypothalamic oxytocin neurons plays a key role in controlling stress-induced social anxiety behaviors.

Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches. [ABSTRACT FROM AUTHOR]

Polycomb group (PcG) proteins are highly conserved proteins assembled into two major types of complexes, PRC1 and PRC2, involved in the epigenetic silencing of a wide range of gene expression programs regulating cell fate and tissue development. The crucial role of PRC1 and PRC2 in the fundamental cellular processes and their involvement in human pathologies such as cancer attracted intense attention over the last few decades. Here, we review recent advancements regarding PRC1 and PRC2 function using the zebrafish model. We point out that the unique characteristics of the zebrafish model provide an exceptional opportunity to increase our knowledge of the role of the PRC1 and PRC2 complexes in tissue development, in the maintenance of organ integrity and in pathology. [ABSTRACT FROM AUTHOR]

Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two asb5 gene isoforms, asb5a and asb5b. Currently, the effects of asb5 gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated asb5a-knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar asb5a and asb5b expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target asb5b in wild-type and asb5a-knockout zebrafish. Knocking down asb5b in the wild-type had no phenotypic impact, but simultaneous asb5b knockdown in asb5a-knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in asb5a and asb5b, with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into asb5’s mechanism.

Pregnenolone (P5) is a steroid that functions in the brain and in zebrafish embryogenesis. It is synthesized from cholesterol via the enzymatic activity of P450scc, encoded by CYP11A1. P5 exerts its function by activating CLIP1, which in turn promotes microtubule assembly necessary for many biological processes including embryogenesis. To examine the functional relatedness of CYP11A1 and CLIP1, we ablated the embryonic expression of both genes in zebrafish, i.e. cyp11a1 and clip1a. Two cyp11a1 knockout fish lines were generated. Both homozygous cyp11a1 knockout lines appeared normal. But the development of fish embryos was delayed and embryonic cell migration was reduced when cyp11a1 function was depleted of by morpholinos. This discrepancy in phenotypes by two different gene depletion methods was also observed for clip1a. While clip1a morphants are defective in embryogenesis, clip1a knockout fish appeared normal. The phenotypes depend on the methods that create gene depletion. While knockout fish lines do not have expected phenotypic defects, clip1a and cyp11a1 morpholinos both reduce embryonic cell migration. We have evaluated the usefulness of both methods of gene ablation, and conclude that CYP11A1 and CLIP1 function in the same pathway to promote embryogenesis. • CYP11A1 and CLIP1 are required for the function of pregnenolone. • Morpholinos against cyp11a1 or clip1a cause defects of zebrafish embryogenesis. • Zebrafish cyp11a1 or clip1a mutants appear normal. • Both morpholinos and CRISPR have limitations, and need to be used carefully. • Cyp11a1 and Clip1a control the same function with the same defects when depleted. [ABSTRACT FROM AUTHOR]

The use of reduced graphene oxide (rGO) and multi‐walled carbon nanotubes (MWCNTs) has attracted considerable interest for fabrication of chemical sensors and biosensors in recent years. In this study, we report on two ultrasensitive DNA biosensors based on a sensing mechanism driven by the electrostatic attraction of [Ru(NH3)6]3+ cations on electrode surfaces modified with Au‐MWCNTs and Au‐rGO. A one‐step pot synthesis was used to prepare these nanocomposites for the development of the DNA‐based biosensors. The detection of DNA with the biosensors was based on the electrostatic attraction of [Ru(NH3)6]3+ cations by the anionic phosphate backbone of DNA. The interfacial changes on the electrode surface upon hybridisation were detected by differential pulse voltammetry (DPV). The high conductivity and high surface to volume ratio properties of the carbon materials enabled significant enhancement of the response for the target DNA detection. The DPV response gave linear concentration ranges (LCRs) of 0.1–10,000 pM and 1–100 pM with the Au‐MWCNT and Au‐rGO‐based biosensors, respectively. Thus, indicating that the MWCNT‐based biosensor was more sensitive for DNA detection and achieved a wider LCR. Nevertheless, both MWCNT and rGO‐based biosensors exhibited very good discrimination against mismatch sequences. [ABSTRACT FROM AUTHOR]

Background: Mutations in lipoxygenase homology domain 1 (LOXHD1) cause autosomal recessive inheritance, leading to high-frequency and intermediatefrequency hearing losses in patients. To date, studies on the localization of LOXHD1 gene expression are limited. In this study, we aimed to observe the expressions of Loxhd1b in zebrafish, C57BL/6 murine cochlea, and HEI-OC1 cells. Methods: The expression of Loxhd1b in the auditory system of zebrafish was explored by in situ hybridization experiments of zebrafish embryos. The expression of Loxhd1b in cochlear and HEI-OC1 cells of C57BL/6 mice was analyzed by immunofluorescence staining. Confocal microscopic in vivo imaging was used to detect the number and morphological characteristics of lateral line neuromasts and inner ear hair cells in zebrafish that knocked down Loxhd1b gene. The effect of knockdown Loxhd1b gene on the development of zebrafish otolith and semicircular canal was observed using microscopic. Transcriptome sequencing was used to identify downstream molecules and associated signaling pathways and validated by western blotting, immunostaining, and rescue experiments. Results: Results of the in situ hybridization with zebrafish embryos at different time points showed that Loxhd1b was expressed in zebrafish at the inner ear and olfactory pores, while the immunostaining showed that Loxhd1 was expressed in both C57BL/6 mouse cochlea and HEI-OC1 cells. Loxhd1b knockdown causes a decrease in the number of spinal and lateral line neuromasts in the inner ear of zebrafish, accompanied by weakened hearing function, and also leads to developmental defects of otoliths and ear follicles. The results of transcriptomics analysis revealed the downstream molecule brain-derived neurotrophic factor (BDNF) and verified that Loxhd1b and BDNF regulate the formation of zebrafish hair cells by synergistic regulation of BDNF/TrkB/ERK pathway based on western blotting, immunostaining, and rescue experiments. Conclusion: This was the first time that the BDNF/TrkB/ERK pathway was identified to play a critical role in the molecular regulation of the development of zebrafish hair cells and the auditory development by Loxhd1b. [ABSTRACT FROM AUTHOR]

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities. [ABSTRACT FROM AUTHOR]

The HCN4 channel is essential for heart rate regulation in vertebrates by generating pacemaker potentials in the sinoatrial node. HCN4 channel abnormality may cause bradycardia and sick sinus syndrome, making it an important target for clinical research and drug discovery. The zebrafish is a popular animal model for cardiovascular research. They are potentially suitable for studying inherited heart diseases, including cardiac arrhythmia. However, it has not been determined how similar the ion channels that underlie cardiac automaticity are in zebrafish and humans. In the case of HCN4, humans have one gene, whereas zebrafish have two ortholog genes (DrHCN4 and DrHCN4L; 'Dr' referring to Danio rerio). However, it is not known whether the two HCN4 channels have different physiological functions and roles in heart rate regulation. In this study, we characterized the biophysical properties of the two zebrafish HCN4 channels in Xenopus oocytes and compared them to those of the human HCN4 channel. We found that they showed different gating properties: DrHCN4L currents showed faster activation kinetics and a more positively shifted G-V curve than did DrHCN4 and human HCN4 currents. We made chimeric channels of DrHCN4 and DrHCN4L and found that cytoplasmic domains were determinants for the faster activation and the positively shifted G-V relationship in DrHCN4L. The use of a dominant-negative HCN4 mutant confirmed that DrHCN4 and DrHCN4L can form a heteromultimeric channel in Xenopus oocytes. Next, we confirmed that both are sensitive to common HCN channel inhibitors/blockers including Cs+, ivabradine, and ZD7288. These HCN inhibitors successfully lowered zebrafish heart rate during early embryonic stages. Finally, we knocked down the HCN4 genes using antisense morpholino and found that knocking down either or both of the HCN4 channels caused a temporal decrease in heart rate and tended to cause pericardial edema. These findings suggest that both DrHCN4 and DrHCN4L play a significant role in zebrafish heart rate regulation. [ABSTRACT FROM AUTHOR]

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms 1/2 (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 messenger RNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression toward a primed pluripotent state. [ABSTRACT FROM AUTHOR]

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5′-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis. [ABSTRACT FROM AUTHOR]

The mechanisms by which tissues and organs achieve their final size and shape during development are largely unknown. Although we have learned much about the mechanisms that control growth, little is known about how those play out to achieve a structure's specific final size and shape. The wings of insects are attractive systems for the study of the control of morphogenesis, because they are perfectly flat and two‐dimensional, composed of two closely appressed cellular monolayers in which morphogenetic processes can be easily visualized. The wings of Lepidoptera arise from imaginal disks whose structure is always perfectly congruent with that of the adult wing, so that it is possible to fate‐map corresponding positions on the larval disk to those of the adult wing. Here we show that the forewing imaginal disks of Junonia coenia are subdivided into four domains, with characteristic patterns of expression of known patterning genes Spalt (Sal), Engrailed (En), and Cubitus interruptus (Ci). We show that DNA and protein synthesis, as well as mitoses, are spatially patterned in a domain‐specific way. Knockdown of Sal and En using produced domain‐specific reductions in the shape of the forewing. Knockdown of signaling pathways involved in the regulation of growth likewise altered the shape of the forewing in a domain‐specific way. Our results reveal a multi‐level regulation of forewing shape involving hormones and growth‐regulating genes. Highlights: The wing imaginal disks of the butterfly, Junonia coenia, are subdivided into four domains, with characteristic patterns of expression of known patterning genes.Cell division is not homogeneous but patterned by the compartments.Disruption of signaling pathways restricts growth in a compartment‐specific manner.Variation in wing shape across the Lepidoptera is due to semi‐independent variation in the relative sizes of the compartments. [ABSTRACT FROM AUTHOR]

The control processes that underlie the progression of development can be summarized in maps of gene regulatory networks (GRNs). A critical step in their assembly is the systematic perturbation of network candidates. In sea urchins the most important method for interfering with expression in a gene-specific way is application of morpholino antisense oligonucleotides (MOs). MOs act by binding to their sequence complement in transcripts resulting in a block in translation or a change in splicing and thus result in a loss of function. Despite the tremendous success of this technology, recent comparisons to mutants generated by genome editing have led to renewed criticism and challenged its reliability. As with all methods based on sequence recognition, MOs are prone to off-target binding that may result in phenotypes that are erroneously ascribed to the loss of the intended target. However, the slow progression of development in sea urchins has enabled extremely detailed studies of gene activity in the embryo. This wealth of knowledge paired with the simplicity of the sea urchin embryo enables careful analysis of MO phenotypes through a variety of methods that do not rely on terminal phenotypes. This article summarizes the use of MOs in probing GRNs and the steps that should be taken to assure their specificity.

A potential treatment for retinal diseases is to induce an endogenous Müller glia (MG)-derived regenerative response to replace damaged neurons. In contrast to mammalian MG, zebrafish MG are capable of mediating spontaneous regeneration. We seek to define the mechanisms that enable retina regeneration in zebrafish in order to identify therapeutic targets to induce mammalian retina regeneration. We previously used pharmacological and genetic methods to inhibit gamma aminobutyric acid A (GABAA) receptors in undamaged zebrafish retinas and showed that such inhibition could induce initiation of retina regeneration, as measured by the dedifferentiation of MG and the appearance of MG-derived proliferating progenitor cells. Here, we show that inhibition of a pharmacologically distinct subset of GABAA receptors (GABAA-ρ) can also induce retina regeneration. Dual inhibition of both GABA receptor subtypes led to enhanced retina regeneration. Gene expression analyses indicate that inhibition of GABAA-ρ receptors induces a canonical retinal regenerative response. Our results support a model in which decreased levels of GABA, such as would occur after retinal cell death or damage, induce dedifferentiation of MG and the generation of proliferating progenitor cells during zebrafish retina regeneration. Animal experiments were approved by the Vanderbilt's Institutional Animal Care and Use Committee (Protocol M1800200) on January 29, 2019.

Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches.

Polycomb group (PcG) proteins are highly conserved proteins assembled into two major types of complexes, PRC1 and PRC2, involved in the epigenetic silencing of a wide range of gene expression programs regulating cell fate and tissue development. The crucial role of PRC1 and PRC2 in the fundamental cellular processes and their involvement in human pathologies such as cancer attracted intense attention over the last few decades. Here, we review recent advancements regarding PRC1 and PRC2 function using the zebrafish model. We point out that the unique characteristics of the zebrafish model provide an exceptional opportunity to increase our knowledge of the role of the PRC1 and PRC2 complexes in tissue development, in the maintenance of organ integrity and in pathology.

The dead end gene has been identified as a essential factor for Primordial germ cells (PGCs) migration and survival in many species, but its role in Monopterus albus is unclear. In order to clarify the function of dead end gene in M.albus PGCs migration and survival, we first characterized the expression profile of M.albus dead end (Madnd) in developing embryos and various tissues. qRT-PCR revealed that Madnd transcripts were exclusively detected in gonad, including ovary, testis and ovotestis.Embryos injected with a Madnd morpholino (Madnd-MO) exhibited down-regulation of the vasa gene. Furthermore, the GFP signal show the PGCs migration in control group were injected with GFP-nanos3 3′-UTR mRNA for visualization, as described in a previous study, yet it was disappeared after embryos injected with Madnd-MO.Finally, we characterized the genomics sequence of the Madnd gene and designed five gRNAs for genome editing. Three gRNAs were selected for microinjection according to the results of in vitro tests. gRNAd1 was used for microinjection with the Cas9 protein and was confirmed to be effective. Our analysis in this study suggested that Madnd play a key role in PGCs migration and survival in M. albus. These data provide the basis for the production of fast-growing and reproductively M.albus sterile. • Expression profile of Madnd gene in developing embryos and tissues was reported. • Madnd was crucial for PGCs migration and survival in embryos. • Genome editing technology was developed for the further studies in the future. [ABSTRACT FROM AUTHOR]

Coagulation factor V (FV) is a liver-derived protein encoded by the F5 gene. Alternative splicing of F5 exon 13 produces a low-abundance splicing isoform, known as FV-short, which binds the anticoagulant protein tissue factor pathway inhibitor (TFPIα) with high affinity, stabilising it in the circulation and potently enhancing its anticoagulant activity. Accordingly, rare F5 gene mutations that up-regulate FV-short splicing are associated with bleeding. In this study we have explored the possibility of decreasing FV-short splicing by antisense-based splicing modulation. To this end, we have designed morpholino antisense oligonucleotides (MAOs) targeting the FV-short-specific donor and acceptor splice sites and tested their efficacy in a liver cell line (HepG2) that naturally expresses full-length FV and FV-short. Cells were treated with 0–20 µM MAO, and full-length FV and FV-short mRNA expression was analysed by RT-(q)PCR. Both MAOs, alone or in combination, decreased the FV-short/full-length FV mRNA ratio down to ~50% of its original value in a specific and dose-dependent manner. This pilot study provides proof-of-principle for the possibility to decrease FV-short expression by antisense-mediated splicing modulation. In turn, this may form the basis for novel therapeutic approaches to bleeding disorders caused by FV-short over-expression and/or elevated TFPIα (activity) levels. [ABSTRACT FROM AUTHOR]

Echinoderms are important experimental models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered developmental studies using these animals. Morpholino antisense oligonucleotides (MOs) have been used successfully by the echinoderm research community for almost two decades, and MOs remain the most widely used tool for acute gene knockdowns in these organisms. Echinoderm embryos develop externally and are optically transparent, making them ideally-suited to many light-based approaches for analyzing and manipulating development. Studies using zebrafish embryos have demonstrated the effectiveness of photoactivatable (caged) MOs for conditional gene knockdowns. Here we show that caged MOs, synthesized using nucleobase-caged monomers, provide light-regulated control over gene expression in sea urchin embryos. Our work provides the first robust approach for conditional gene silencing in this prominent model system. • Caged morpholinos are highly effective in sea urchin embryos. • Conditional gene silencing can be used to study mechanisms of echinoderm development. • Caged morpholinos will allow high-resolution dissection of developmental gene regulatory networks. [ABSTRACT FROM AUTHOR]

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.

Oxygen uptake (ṀO2) in larval zebrafish prior to maturation of the gill relies on cutaneous O2 transfer. Under normoxic conditions, rates of cutaneous O2 transfer are unaffected by haemoglobin availability but are diminished in fish lacking a functional circulatory system, suggesting that internal convection is critically involved in setting the resting ṀO2 in zebrafish larvae, even when relying on cutaneous O2 transfer. The reliance of ṀO2 on blood circulation led to the first objective of the current study, to determine whether loss of internal convection would reduce acute hypoxia performance (as determined by measuring critical PO2; Pcrit) in larval zebrafish under conditions of moderate hypoxia (PO2 = 55 mmHg) at 28.5 and 34 °C. Internal convection was eliminated by preventing development of blood vessels using morpholino knockdown of vascular endothelial growth factor (VEGF); these fish are termed VEGF morphants. Breathing frequency (fV) and heart rate (fH) also were measured (at 28.5 °C) to determine whether any detriment in performance might be linked to cardiorespiratory dysfunction. Although ṀO2 was reduced in the VEGF morphants, there was no significant effect on Pcrit at 28.5 °C. Raising temperature to 34 °C resulted in the VEGF morphants exhibiting a higher Pcrit than the shams, suggesting an impairment of hypoxia tolerance in the morphants at the higher temperature. The usual robust increase in fV during hypoxia was absent or attenuated in VEGF morphants at 4 and 5 days post fertilization (dpf), respectively. Resting fH was reduced in the VEGF morphants and unlike the sham fish, the morphants did not exhibit hypoxic tachycardia at 4 or 5 dpf. The number of cutaneous neuroepithelial cells (presumptive O2 chemoreceptors) was significantly higher in the VEGF morphants and thus the cardiorespiratory impairment in the morphants during hypoxia was unlikely related to inadequate peripheral O2 sensing. [ABSTRACT FROM AUTHOR]

The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization. • Morpholino-peptide based oligomers show a preferred secondary structure. • Morpholino-glycine based oligomers form complexes with DNA in a 2: 1 molar ratio. • The complex with DNA is stabilized by hydrophobic contacts among bases and H-bonds between the peptide backbone. [ABSTRACT FROM AUTHOR]

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5′-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis.

LIM homeobox 9 (Lhx9) is a member of the LIM homeodomain transcription factor family, which expresses and functions in various vertebrate tissues, such as the gonads and pineal gland. Previous studies on lhx9 in zebrafish have mainly focused on the brain. However, little is known about the expression pattern of lhx9 during embryogenesis. Here, we detected lhx9 expression in zebrafish embryos using whole-mount in situ hybridization and found lhx9 expressed in heart, pectoral fin, and retina during their development in zebrafish. We then detailed the expression of lhx9 in retinal development. To further investigate the function of Lhx9 in retinogenesis, we performed morpholino (MO) knockdown experiments and found that upon lhx9 knockdown by MO, larvae presented normal eye development, retinal neural development, differentiation, proliferation, apoptosis, and responses to light stimulus. We not only elaborated the expression pattern of lhx9 in zebrafish embryogenesis, but we also demonstrated that lhx9 knockdown by morpholino does not affect the zebrafish retinal development, and our study provides data for further understanding of the role of Lhx9 in zebrafish retinal development.

The Enteric Nervous System (ENS) is a large network of enteric neurons and glia that regulates various processes in the gastrointestinal tract including motility, local blood flow, mucosal transport and secretion. The ENS is derived from stem cells coming from the neural crest that migrate into and along the primitive gut. Defects in ENS establishment cause enteric neuropathies, including Hirschsprung disease (HSCR), which is characterized by an absence of enteric neural crest cells in the distal part of the colon. In this review, we discuss the use of zebrafish as a model organism to study the development of the ENS. The accessibility of the rapidly developing gut in zebrafish embryos and larvae, enables in vivo visualization of ENS development, peristalsis and gut transit. These properties make the zebrafish a highly suitable model to bring new insights into ENS development, as well as in HSCR pathogenesis. Zebrafish have already proven fruitful in studying ENS functionality and in the validation of novel HSCR risk genes. With the rapid advancements in gene editing techniques and their unique properties, research using zebrafish as a disease model, will further increase our understanding on the genetics underlying HSCR, as well as possible treatment options for this disease.

Cilia are highly conserved organelles present in almost all types of eukaryotic cells, and defects in cilia structure and/or function are related to many human genetic disorders. Single-celled ciliated protists, which possess diverse types of cilia, are remarkable model organisms for studying cilia structures and functions. Euplotes vannus is a representative ciliate with many intriguing features; for example, it possesses extensively fragmented somatic genomes and a high frequency of + 1 programmed ribosomal frameshifting. However, the molecular mechanisms underlying these remarkable traits remain largely unknown, mainly due to the lack of efficient genetic manipulation tools. Here, we describe the first application of a morpholino-based strategy to knockdown gene expression in E. vannus. Through interfering with the function of two ciliary genes, ZMYND10 and C21ORF59, we showed that these two genes are essential for the ciliary motility and proliferation of E. vannus cells. Strikingly, both ZMYND10- and C21ORF59-knockdown cells developed shorter cilia in the ventral cirri, a special type of ciliary tuft, suggesting a novel role for these genes in the regulation of cilia length. Our data provide a new method to explore gene function in E. vannus, which may help us to understand the functions of evolutionarily conserved cilia-related genes as well as other biological processes in this intriguing model.

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration., Competing Interests: Conflict of interest The authors declare no conflict of interest.

Transcription factors that contain a homeodomain DNA-binding domain have crucial functions in most aspects of cellular function and embryonic development in both animals and plants. Hmx proteins are a subfamily of NK homeodomain-containing proteins that have fundamental roles in development of sensory structures such as the eye and the ear. However, Hmx functions in spinal cord development have not been analyzed. Here, we show that zebrafish (Danio rerio) hmx2 and hmx3a are coexpressed in spinal dI2 and V1 interneurons, whereas hmx3b, hmx1, and hmx4 are not expressed in spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously reported role in ear development, hmx3a is required for correct specification of a subset of spinal interneuron neurotransmitter phenotypes, as well as correct lateral line progression and survival to adulthood. Surprisingly, despite similar expression patterns of hmx2 and hmx3a during embryonic development, zebrafish hmx2 mutants are viable and have no obviously abnormal phenotypes in sensory structures or neurons that require hmx3a. In addition, embryos homozygous for deletions of both hmx2 and hmx3a have identical phenotypes to severe hmx3a single mutants. However, mutating hmx2 in hypomorphic hmx3a mutants that usually develop normally, results in abnormal ear and lateral line phenotypes. This suggests that while hmx2 cannot compensate for loss of hmx3a, it does function in these developmental processes, although to a much lesser extent than hmx3a. More surprisingly, our mutational analyses suggest that Hmx3a may not require its homeodomain DNA-binding domain for its roles in viability or embryonic development. [ABSTRACT FROM AUTHOR]

Cilia are highly conserved organelles present in almost all types of eukaryotic cells, and defects in cilia structure and/or function are related to many human genetic disorders. Single-celled ciliated protists, which possess diverse types of cilia, are remarkable model organisms for studying cilia structures and functions. Euplotes vannus is a representative ciliate with many intriguing features; for example, it possesses extensively fragmented somatic genomes and a high frequency of + 1 programmed ribosomal frameshifting. However, the molecular mechanisms underlying these remarkable traits remain largely unknown, mainly due to the lack of efficient genetic manipulation tools. Here, we describe the first application of a morpholino-based strategy to knockdown gene expression in E. vannus. Through interfering with the function of two ciliary genes, ZMYND10 and C21ORF59 , we showed that these two genes are essential for the ciliary motility and proliferation of E. vannus cells. Strikingly, both ZMYND10- and C21ORF59-knockdown cells developed shorter cilia in the ventral cirri, a special type of ciliary tuft, suggesting a novel role for these genes in the regulation of cilia length. Our data provide a new method to explore gene function in E. vannus , which may help us to understand the functions of evolutionarily conserved cilia-related genes as well as other biological processes in this intriguing model. [ABSTRACT FROM AUTHOR]

Angiopoietin/TIE signalling plays a major role in blood and lymphatic vessel development. In mouse, Tek (previously known as Tie2) mutants die prenatally due to a severely underdeveloped cardiovascular system. In contrast, in zebrafish, previous studies have reported that although embryos injected with tek morpholinos (MOs) exhibit severe vascular defects, tek mutants display no obvious vascular malformations. To further investigate the function of zebrafish Tek, we generated a panel of loss-of-function tek mutants, including RNA-less alleles, an allele lacking the MO-binding site, an in-frame deletion allele and a premature termination codon-containing allele. Our data show that all these mutants survive to adulthood with no obvious cardiovascular defects. MO injections into tek mutants lacking the MO-binding site or the entire tek locus cause similar vascular defects to those observed in MO-injected +/+ siblings, indicating off-target effects of the MOs. Surprisingly, comprehensive phylogenetic profiling and synteny analyses reveal that Tek was lost in the largest teleost clade, suggesting a lineage-specific shift in the function of TEK during vertebrate evolution. Altogether, these data show that Tek is dispensable for zebrafish development, and probably dispensable in most teleost species. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Coagulation factor V (FV) is a liver-derived protein encoded by the F5 gene. Alternative splicing of F5 exon 13 produces a low-abundance splicing isoform, known as FV-short, which binds the anticoagulant protein tissue factor pathway inhibitor (TFPIα) with high affinity, stabilising it in the circulation and potently enhancing its anticoagulant activity. Accordingly, rare F5 gene mutations that up-regulate FV-short splicing are associated with bleeding. In this study we have explored the possibility of decreasing FV-short splicing by antisense-based splicing modulation. To this end, we have designed morpholino antisense oligonucleotides (MAOs) targeting the FV-short-specific donor and acceptor splice sites and tested their efficacy in a liver cell line (HepG2) that naturally expresses full-length FV and FV-short. Cells were treated with 0–20 µM MAO, and full-length FV and FV-short mRNA expression was analysed by RT-(q)PCR. Both MAOs, alone or in combination, decreased the FV-short/full-length FV mRNA ratio down to ~50% of its original value in a specific and dose-dependent manner. This pilot study provides proof-of-principle for the possibility to decrease FV-short expression by antisense-mediated splicing modulation. In turn, this may form the basis for novel therapeutic approaches to bleeding disorders caused by FV-short over-expression and/or elevated TFPIα (activity) levels.

The TGF-β superfamily comprises two distinct branches: the Activin/ Nodal and BMP pathways. During development, signaling by this superfamily regulates a variety of embryological processes, and it has a conserved role in patterning the dorsal-ventral body axis. Recent studies show that BMP signaling establishes the dorsal-ventral axis in some mollusks. However, previous pharmacological inhibition studies in the annelid Capitella teleta, a sister clade to the mollusks, suggests that the dorsal-ventral axis is patterned via Activin/Nodal signaling. Here, we determine the role of both the Activin/Nodal and BMP pathways as they function in Capitella axis patterning. Antisense morpholino oligonucleotides were targeted to Ct-Smad2/3 and Ct-Smad1/5/8, transcription factors specific to the Activin/Nodal and BMP pathways, respectively. Following microinjection of zygotes, resulting morphant larvae were scored for axial anomalies. We demonstrate that the Activin/Nodal pathway of the TGF-β superfamily, but not the BMP pathway, is the primary dorsal-ventral patterning signal in Capitella. These results demonstrate variation in the molecular control of axis patterning across spiralians, despite sharing a conserved cleavage program. We suggest that these findings represent an example of developmental system drift. [ABSTRACT FROM AUTHOR]

The transcription factor Pax6 is an important regulator of early animal development. Loss of function mutations of pax6 in a range of animals result in a reduction or complete loss of the eye, a reduction of a subset of neurons, and defects in axon growth. There are no studies focusing on the role of pax6 during development of any lophotrochozoan representative, however, expression of pax6 in the developing eye and nervous system in a number of species suggest that pax6 plays a highly conserved role in eye and nervous system formation. We investigated the functional role of pax6 during development of the marine annelid Capitella teleta. Expression of pax6 transcripts in C. teleta larvae is similar to patterns found in other animals, with distinct subdomains in the brain and ventral nerve cord as well as in the larval and juvenile eye. To perturb pax6 function, two different splice-blocking morpholinos and a translation-blocking morpholino were used. Larvae resulting from microinjections with either splice-blocking morpholino show a reduction of the pax6 transcript. Development of both the larval eyes and the central nervous system architecture are highly disrupted following microinjection of each of the three morpholinos. The less severe phenotype observed when only the homeodomain is disrupted suggests that presence of the paired domain is sufficient for partial function of the Pax6 protein. Preliminary downstream target analysis confirms disruption in expression of some components of the retinal gene regulatory network, as well as disruption of genes involved in nervous system development. Results from this study, taken together with studies from other species, reveal an evolutionarily conserved role for pax6 in eye and neural specification and development. • pax6 is expressed during eye and nervous system development in the annelid Capitella. • Morpholino knockdown of pax6 results in central nervous system abnormalities. • Eye photoreceptor and pigment cell formation are disrupted in pax6 morphants. • Paired domain alone is sufficient for partial Pax6function. [ABSTRACT FROM AUTHOR]

There is an ongoing effort to increase the efficiency of gene delivery for the regulation of diseases-related genes. In this report, we demonstrate the efficiency of a DNA-based nanostructure to deliver morpholino antisense oligonucleotides for the upregulated genes in breast cancer as an alternative to the currently used delivery systems. A DNA-tile structure is constructed by embedding antisense oligonucleotides targeting the HER2 and ERα genes. Then, the sticky ends of the DNA-tile nanostructures are hybridized to gold nanoparticles (AuNPs) coated with the complementary oligonucleotides to enhance their cellular uptake. It is found that the morpholino antisense oligonucleotide embedded DNA-tile-AuNPs structure is 30% more effective than the liposome-based system to deliver morpholinos and induce gene silencing in breast cancer cells. The results of the study suggest that the prepared novel nanostructure is an effective and biocompatible carrier that can be used in in vitro gene silencing studies and can be further pursued in in vivo studies. [ABSTRACT FROM AUTHOR]

Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is a member of centromere protein family that regulates chromosome segregation during mitosis. However, its necessity in early embryo development has not been fully investigated. In this study, expression and function of CENPF was investigated in mouse early embryogenesis. Detection of CENPF expression and localization revealed a cytoplasm, spindle and nuclear membrane related dynamic pattern throughout mitotic progression. Farnesyltransferase inhibitor (FTI) was employed to inhibit CENPF farnesylation in zygotes. The results showed that CENPF degradation was inhibited and its specific localization on nuclear membranes in morula and blastocyst vanished after FTI treatment. Also, CAAX motif mutation leads to failure of CENPF-C630 localization in morula and blastocyst. These results indicate that farnesylation plays a key role during CENPF degradation and localization in early embryos. To further assess CENPF function in parthenogenetic or fertilized embryos development, morpholino (MO) and Trim-Away were used to disturb CENPF function. CENPF knockdown in Metaphase II (MII) oocytes, zygotes or embryos with MO approach resulted in failure to develop into morulae and blastocysts, revealing its indispensable role in both parthenogenetic and fertilized embryos. Disturbing of CENPF with Trim-Away approach in zygotes resulted in impaired development of 2-cell and 4-cell, but did not affect the morula and blastocyst formation because of the recovered expression of CENPF. Taken together, our data suggest CENPF plays an important role during early embryonic development in mice. Abbreviation: CENPF: centromere protein F; MO: morpholino; FTI: Farnesyltransferase inhibitor; CENPE: centromere protein E; IVF: in vitro fertilization; MII: metaphase II; SAC: spindle assembly checkpoint; Mad1: mitotic arrest deficient 1; BUB1: budding uninhibited by benzimidazole 1; BUBR1: BUB1 mitotic checkpoint serine/threonine kinase B; Cdc20: cell division cycle 20 [ABSTRACT FROM AUTHOR]

The phenotypes caused by morpholino-mediated interference of gene function in zebrafish are often not observed in the corresponding mutant(s). We took advantage of the availability of a relatively large collection of transcriptomic datasets to identify common signatures that characterize morpholino-injected animals (morphants). In addition to the previously reported activation of tp53 expression, we observed increased expression of the interferon-stimulated genes (ISGs), isg15 and isg20 , the cell death pathway gene casp8 , and other cellular stress response genes including phlda3 , mdm2 and gadd45aa. Studies involving segmentation stage embryos were more likely to show upregulation of these genes. We also found that the expression of these genes could be upregulated by increasing doses of an egfl7 morpholino, or even high doses of the standard control morpholino. Thus, these data show that morpholinos can induce the expression of ISGs in zebrafish embryos and further our understanding of morpholino effects. • Machine learning revealed isg and tp53 induction in zebrafish morphants. • Increase in isg and tp53 expression in morphants is highest at segmentation stages. • isg expression is upregulated by increasing doses of some morpholinos. [ABSTRACT FROM AUTHOR]

To address the patterning function of the Bmp2, Bmp4 and Bmp7 growth factors, we designed antisense morpholino oligomers (MO) that block their activity in Xenopus laevis. Bmp4 knockdown was sufficient to rescue the ventralizing effects caused by loss of Chordin activity. Double Bmp4 and Bmp7 knockdown inhibited tail development. Triple Bmp2/Bmp4/Bmp7 depletion further compromised trunk development but did not eliminate dorsoventral patterning. Unexpectedly, we found that blocking Spemann organizer formation by UV treatment or beta-Catenin depletion caused BMP inhibition to have much more potent effects, abolishing all ventral development and resulting in embryos having radial central nervous system (CNS) structures. Surprisingly, dorsal signaling molecules such as Chordin, Noggin, Xnr6 and Cerberus were not re-expressed in these embryos. We conclude that BMP inhibition is sufficient for neural induction in vivo, and that in the absence of ventral BMPs, Spemann organizer signals are not required for brain formation.

The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.

Paralysis following spinal cord injury (SCI) is due to failure of axonal regeneration. It is believed that the capacities of neurons to regrow their axons are due partly to their intrinsic characteristics, which in turn are greatly influenced by several types of inhibitory molecules that are present, or even increased in the extracellular environment of the injured spinal cord. Many of these inhibitory molecules have been studied extensively in recent years. It has been suggested that the small GTPase RhoA is an intracellular convergence point for signaling by these extracellular inhibitory molecules, but due to the complexity of the central nervous system (CNS) in mammals, and the limitation of pharmacological tools, the specific roles of RhoA are unclear. By exploiting the anatomical and technical advantages of the lamprey CNS, we recently demonstrated that RhoA knockdown promotes true axon regeneration through the lesion site after SCI. In addition, we found that RhoA knockdown protects the large, identified reticulospinal neurons from apoptosis after their axons were axotomized in spinal cord. Therefore, manipulation of the RhoA signaling pathway may be an important approach in the development of treatments that are both neuroprotective and axon regeneration-promoting, to enhance functional recovery after SCI.

Morpholino-containing aza-BODIPYs at 3,5-positions were synthesized. The maxima absorption and emission of these dyes locate at the near-infrared region. Aza-BODIPY 1 with the morpholino group as a pH-sensitive functionality could be used to be a pH probe, and the dramatic increase in fluorescence intensity at 675 nm by about 1500 folds. Moreover, the singlet oxygen generation of PS 2 with the dibromo groups at 2,6-positions was more effective than that of the parent dye 1. Image 1 • Morpholino-containing aza-BODIPYs at 3,5-positions were synthesized. • Aza-BODIPY 1 with the morpholino group as a pH-sensitive functionality could be used to be a pH probe. • The singlet oxygen generation of PS 2 was more effective than that of the parent dye 1. [ABSTRACT FROM AUTHOR]

Morpholinos are six‐membered rings that may provide higher conformational rigidity when incorporated into an oligonucleotide (ODN) backbone. Phosphorodiamidate morpholinos are chemically modified ODNs containing morpholinos in place of 2'‐deoxyribose moieties throughout their backbone and have garnered much interest in recent years due to their ability to function as highly effective steric blockers in exon skipping therapy. To further explore the biophysical and biological properties of ODNs derived from morpholino nucleosides, we have replaced the 2'‐deoxyribonucleotides of phosphodiester DNA with morpholinonucleotides to generate phosphoramidate ODNs. Here, we evaluate the mechanistic pathways observed during the solution‐phase synthesis of morpholinonucleoside phosphoramidites, solid‐phase synthesis of morpholinonucleotide phosphoramidates of mA, mG, mC and mT (prefix 'm' represents morpholino) and our first attempts directed at the solid‐phase synthesis of chimeric DNA‐phosphoramidate ODNs, as well as fully modified 22‐mer phosphoramidate ODNs. [ABSTRACT FROM AUTHOR]