Antisense Morpholino Research Articles

The Spliceosomal Component Sf3b1is Essential for Hematopoietic Stem Cell Formation through the Regulation of the Jak/Stat Signaling Pathway

Hematopoietic stem cells (HSCs) maintain multi-lineage hematopoiesis for the lifetime of an organism. During embryogenesis, HSCs first emerge from specialized hemogenic endothelial cells within the dorsal aorta through a process termed the endothelial-to-hematopoietic transition. While much is known about the role of transcription factors and transcriptional control in HSC specification, it is ill defined how post-transcriptional RNA processing influences this first HSC fate decision. Using zebrafish loss-of-function mutants for the pre-mRNA spliceosomal component sf3b1 (splicing factor 3b, subunit 1), we demonstrated that impairing splicing hindered HSC production, specifically at the endothelial-to-hematopoietic transition. Loss-of-function sf3b1hi3394 mutants have diminished expression of thehemogenic endothelial and HSC markers runx1 and gata2b, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf (hours post fertilization). These data indicate that although sf3b1 is ubiquitously expressed, hemogenic endothelium and the downstream HSCs are more sensitive to its loss than other endothelial cells. To uncover the mechanism underlying the defect in hemogenic endothelium and HSCs in sf3b1 mutants, we performed RNA-sequencing on purified kdrl:gfp- positiveendothelial cells from sf3b1 mutants and wild-type siblings at 24 hpf. We observed nearly 900 genes were mis-spliced and of those only 144 were differentially expressed. This group was significantly enriched for genes involved in mRNA processing and Jak (Janus kinase)/Stat (Signaling transducer and activator of transcription) signaling. In particular, target genes of the Jak/Stat pathway were significantly enriched in the downregulated gene list and many components of the signaling pathway were mis-spliced including il6st (interleukin 6 signal transducer), the gene encoding the IL6-family common receptor Gp130 (Glycoprotein 130).To determine if mis-splicing of il6st was sufficient to decrease HSC production, we injected antisense morpholinos into wild-type zebrafish to mimic the sf3b1 mutant - specific exon-skipping event in il6st and found significantly diminished HSC levels at 28 hpf. Stat3 is the major transcription factor downstream of Gp130, thus we ubiquitously overexpressed a constitutively-active form of Stat3 and found it could significantly suppress the HSC defects in sf3b1 mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. Mutations in SF3B1 are prevalent in myelodysplastic syndrome (MDS), a disorder originating in HSC dysfunction. Altered STAT3 signaling is also common in MDS, thus our data suggest that SF3B1 disruptions in HSCs could be contributing to MDS via mis-regulation of JAK/STAT signaling. DisclosuresNo relevant conflicts of interest to declare.

ETV6/TEL1 Is Required for HSC Emergence during Embryonic Development.

ETV6/TEL1 (hereinafter referred to as TEL1) is an ets family transcription factor that is frequently involved in chromosomal translocations associated with leukaemia, including t(12:21) which generates the TEL-AML1 fusion gene, the most common genetic abnormality in childhood cancer. The role of TEL1 in normal haemopoiesis has been extensively studied in the mouse where knock-out studies have indicated that TEL1 is not required for development of primitive (ie yolk sac-derived) haemopoiesis. TEL1-knockout mice die at day E10.5 due to a defect in angiogenesis precluding direct analysis of the definitive (ie intraembryonically-derived) haematopoietic system. Analysis of chimaeric mice generated using TEL1−/− ES cells suggest that TEL1 is not required for generation of the definitive haemopoietic system during ontogeny but is required for HSC survival perinatally at bone marrow stages. Conditional knockout studies have subsequently identified a role for TEL1 in the survival of adult bone marrow-derived HSC. We now report the analysis of TEL1 function in amphibian (Xenopus) embryos where haemopoietic development can be studied even in embryos with defective angiogenesis. We characterised the TEL1 expression pattern and knocked down its function using antisense morpholino oligonucleotide injection. Consistent with the mouse knockout, specification of the primitive haemopoietic system was unaffected, however a defect in maturation of primitive erythroid cells, not previously reported in the mouse, was also apparent. We next analysed the development of the definitive haemopoietic system, thought to arise as clusters of HSCs in the floor of the dorsal aorta. But, no clusters of HSCs were found. Formation of the dorsal aorta however, proceeded normally, indicating that TEL1 controls the decision between the arterial and HSC programmes. Given, the critical role for TEL1 in the development of HSC indicated by these studies, a temporal analysis of emergence of intraembryonic HSC in TEL-deficient mice may be warranted.

Donepezil Reduces Amyloid Precursor Protein Endocytosis by Resulting from Increase in the Expression of Sorting Nexin Protein 33

Donepezil, the most widely used drug for the treatment of Alzheimer's disease (AD), is an acetylcholinesterase (AChE) inhibitor and is thought to improve cognition by stimulating cholinergic neurotransmission. However, no correlation has yet been established between the inhibitory role of AChE inhibitors and their therapeutic effects when used in AD patients. The cleavage pathway of amyloid precursor protein (APP) includes amyloidgenic (β, γ-cleavage) and non-amyloidgenic (α-cleavage) pathways. The intracellular transportation of APP is important in determining these cleavage pathways. It has been suggested that sorting nexin (SNX) family proteins regulates the intracellular transport of APP, thereby enhancing α-cleavage. In this study, we examined the effects of donepezil on SNX33 expression changes and APP processing in primary cultures of fetal rat cortical neurons. While donepezil treatment increased the levels of SNX33 expression and soluble APPα (sAPPα) in culture media, no changes were observed regarding full-length APP expression in the cell lysate. Donepezil also reduced the release of amyloid β (Aβ) into culture media in a concentration- and time-dependent manner. This reduction was not affected by acetylcholine receptor antagonists. The membrane surface expression of APP was elevated by donepezil. Furthermore, SNX knockdown by antisense morpholino oligos prevented the effects of donepezil. These results indicated that donepezil increased APP expression at the surface of the plasma membrane by decreasing APP endocytosis through upregulation of SNX33, suggesting donepezil might stimulate the non-amyloidogenic pathway. This new mechanism of action for the currently used anti-AD drug may provide a valuable basis for future drug discovery.

Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 h u2787 Model of Fragile X Syndrome.

Zebrafish represent a valuable model for investigating the molecular and cellular basis of Fragile X syndrome (FXS). Reduced expression of the zebrafish FMR1 orthologous gene, fmr1, causes developmental and behavioural phenotypes related to FXS. Zebrafish homozygous for the hu2787 non-sense mutation allele of fmr1 are widely used to model FXS, although FXS-relevant phenotypes seen from morpholino antisense oligonucleotide (morpholino) suppression of fmr1 transcript translation were not observed when hu2787 was first described. The subsequent discovery of transcriptional adaptation (a form of genetic compensation), whereby mutations causing non-sense-mediated decay of transcripts can drive compensatory upregulation of homologous transcripts independent of protein feedback loops, suggested an explanation for the differences reported. We examined the whole-embryo transcriptome effects of homozygosity for fmr1hu2787 at 2 days post fertilisation. We observed statistically significant changes in expression of a number of gene transcripts, but none from genes showing sequence homology to fmr1. Enrichment testing of differentially expressed genes implied effects on lysosome function and glycosphingolipid biosynthesis. The majority of the differentially expressed genes are located, like fmr1, on Chromosome 14. Quantitative PCR tests did not support that this was artefactual due to changes in relative chromosome abundance. Enrichment testing of the “leading edge” differentially expressed genes from Chromosome 14 revealed that their co-location on this chromosome may be associated with roles in brain development and function. The differential expression of functionally related genes due to mutation of fmr1, and located on the same chromosome as fmr1, is consistent with R.A. Fisher’s assertion that the selective advantage of co-segregation of particular combinations of alleles of genes will favour, during evolution, chromosomal rearrangements that place them in linkage disequilibrium on the same chromosome. However, we cannot exclude that the apparent differential expression of genes on Chromosome 14 genes was, (if only in part), caused by differences between the expression of alleles of genes unrelated to the effects of the fmr1hu2787 mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.

The cytokine FAM3B/PANDER is an FGFR ligand that promotes posterior development in Xenopus

Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling plays a crucial role in anterior-posterior (A-P) axial patterning of vertebrate embryos by promoting posterior development. In our screens for novel developmental regulators in Xenopus embryos, we identified Fam3b as a secreted factor regulated in ectodermal explants. Family with sequence similarity 3 member B (FAM3B)/PANDER (pancreatic-derived factor) is a cytokine involved in glucose metabolism, type 2 diabetes, and cancer in mammals. However, the molecular mechanism of FAM3B action in these processes remains poorly understood, largely because its receptor is still unidentified. Here we uncover an unexpected role of FAM3B acting as a FGF receptor (FGFR) ligand in Xenopus embryos. fam3b messenger RNA (mRNA) is initially expressed maternally and uniformly in the early Xenopus embryo and then in the epidermis at neurula stages. Overexpression of Xenopus fam3b mRNA inhibited cephalic structures and induced ectopic tail-like structures. Recombinant human FAM3B protein was purified readily from transfected tissue culture cells and, when injected into the blastocoele cavity, also caused outgrowth of tail-like structures at the expense of anterior structures, indicating FGF-like activity. Depletion of fam3b by specific antisense morpholino oligonucleotides in Xenopus resulted in macrocephaly in tailbud tadpoles, rescuable by FAM3B protein. Mechanistically, FAM3B protein bound to FGFR and activated the downstream ERK signaling in an FGFR-dependent manner. In Xenopus embryos, FGFR activity was required epistatically downstream of Fam3b to mediate its promotion of posterior cell fates. Our findings define a FAM3B/FGFR/ERK-signaling pathway that is required for axial patterning in Xenopus embryos and may provide molecular insights into FAM3B-associated human diseases.

RNA‐binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification

Neural crest (NC) cells are a multipotent cell population, unique to the vertebrate species, which gather at the dorsal neural tube and undergo an epithelial-to-mesenchymal transition (EMT). NC then migrate throughout the developing embryo contributing to various cell types such as craniofacial cartilage, neurons, and elements of the peripheral nervous system. While a gene regulatory network (GRN) underlies establishment of NC formation, recent studies suggest a role for post-transcriptional regulation in modulating the output of these regulatory circuits. In this study, we examined RNA-sequencing data from avian embryos at different stages of NC development and found enrichment of Elav1, a transcript encoding for the RNA-binding protein HuR. Using immunohistochemical analyses we characterized the spatiotemporal expression of HuR. We observed HuR expression following the establishment of the neural plate border and upon closure of the neural tube, with an enrichment in expression coinciding with NC formation. Using antisense morpholino oligo to perturb HuR expression, we observed loss of early neural crest specifier FoxD3 and its activator Axud1, as well as the EMT regulator Draxin. In addition, we identified several putative HuR binding sites within the predicted secondary structure of Draxin 3’UTR. RNA pulldown further showed Draxin is a specific target of HuR. Importantly, overexpression of exogenous Draxin was able to rescue NC specification defects observed in HuR knockdowns. Our results demonstrate a critical role for HuR in maintenance of cranial NC specification, at least partially via Draxin mRNA stabilization. Together, these data highlight an important intersection of post-transcriptional regulation with modulation of the neural crest specification GRN.

Anisomycin induces hair cell death and blocks supporting cell proliferation in zebrafish lateral line neuromast

Ototoxicity of drugs is an important inducement for hearing loss. Anisomycin is a candidate drug for parasite, cancer, immunosuppression, and mental disease. However, the ototoxicity of anisomycin has not been examined. In this study, the ototoxicity of anisomycin was evaluated using zebrafish lateral line. We found the zebrafish treated with anisomycin during lateral line development could inhibit hair cell formation in a time- and dose-dependent manner. After neuromasts are mature with differentiated hair cells by 5 day post-fertilization, anisomycin could induce hair cell loss effectively through chronic exposure rather than acute exposure. TUNEL assay and qPCR of apoptosis related genes tp53, casp8, casp3a, and casp3b indicated that cell apoptotic was induced by chronic anisomycin exposure. Furthermore, knocking down tp53 with antisense morpholino could attenuate the hair cell loss induced by anisomycin. In addition, we found that anisomycin chronic exposure also inhibited the proliferation of supporting cell. Together, these results indicate that chronic anisomycin exposure could induce hair cell death and block supporting cell proliferation, which causes hair cell loss in zebrafish neuromast. This study provides primary ototoxicity evaluation for anisomycin.

Functional Validation of a Pathogenic Missense Variant in Human Filamin C Cardiomyopathy through Disruption of a Zebrafish Homologue Recapitulates Cardiac Disease

Purpose We recently performed whole-genome sequencing (WGS) of an East Asian proband and family members with diverse cardiac diseases, including arrhythmias, cardiomyopathies, premature heart failure requiring transplant, and identified a pathogenic missense variant (p.G2151S) in exon 39 of the filamin C gene, FLNC. In this study, we aim to demonstrate recapitulation of the pathogenic effects of the variant using a zebrafish (Danio rerio) model. Methods The proband, 3 other affected and 3 unaffected family members of a four-generation nonconsanguineous Hong Kong Chinese pedigree of 25 individuals were sequenced by WGS with 30X coverage. Single nucleotide variants and insertion-deletions that met our inclusion criteria were shortlisted, and bioinformatic analysis and variant prioritization were performed using Kaviar, gnomAD and dbNSFP databases to identify damaging variants in known cardiac-specific genes. The study was approved by the university/hospital's institutional review board. Sequence alignment of the pathogenic gene region with the zebrafish homologue was performed using the GENESTREAM network server, Institut de Genetique Humaine, Montpellier, France. Knockdown of the region was carried out using microinjection of morpholino oligonucleotides (MO), or control oligomers. Embryonic lethality, survival after birth, animal and cardiac morphology, heart rate, and histopathology were analyzed. Results Using a zebrafish model, we performed sequence alignment and identified the corresponding point mutation in flnca and flncb. Knockdown of the homologous sequences using antisense morpholino oligonucleotides resulted in stunted growth and development of the animal, cardiomegaly, pericardial edema, dose-dependent increase in embryonic lethality (survival rates of 95.8%, 46.6% and 9.7% at day 3 post-fertilization for control oligomer (n=24), 0.1 ng MO (n=88) and 0.5 ng MO (n=154), respectively), and reduced heart rate (134, 108 and 103 beats per minute, respectively; P Conclusion Damaging effects of the missense variant in human filamin C cardiomyopathy have been confirmed in the homologous region in this zebrafish model. The variable phenotypic manifestations within this East Asian family remain to be elucidated.

A targeted antisense therapeutic approach for Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder characterized by premature death from myocardial infarction or stroke. It is caused by de novo single-nucleotide mutations in the LMNA gene that activate a cryptic splice donor site, resulting in the production of a toxic form of lamin A, which is termed progerin. Here we present a potential genetic therapeutic strategy that utilizes antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to block pathogenic splicing of mutant transcripts. Of several candidates, PPMO SRP-2001 provided the most significant decrease in progerin transcripts in patient fibroblasts. Intravenous delivery of SRP-2001 to a transgenic mouse model of HGPS produced significant reduction of progerin transcripts in the aorta, a particularly critical target tissue in HGPS. Long-term continuous treatment with SRP-2001 yielded a 61.6% increase in lifespan and rescue of vascular smooth muscle cell loss in large arteries. These results provide a rationale for proceeding to human trials.

Using antisense oligonucleotides for the physiological modulation of the alternative splicing of NF1 exon 23a during PC12 neuronal differentiation

Neurofibromatosis Type 1 (NF1) is a genetic condition affecting approximately 1:3500 persons worldwide. The NF1 gene codes for neurofibromin protein, a GTPase activating protein (GAP) and a negative regulator of RAS. The NF1 gene undergoes alternative splicing of exon 23a (E23a) that codes for 21 amino acids placed at the center of the GAP related domain (GRD). E23a-containing type II neurofibromin exhibits a weaker Ras-GAP activity compared to E23a-less type I isoform. Exon E23a has been related with the cognitive impairment present in NF1 individuals. We designed antisense Phosphorodiamidate Morpholino Oligomers (PMOs) to modulate E23a alternative splicing at physiological conditions of gene expression and tested their impact during PC12 cell line neuronal differentiation. Results show that any dynamic modification of the natural ratio between type I and type II isoforms disturbed neuronal differentiation, altering the proper formation of neurites and deregulating both the MAPK/ERK and cAMP/PKA signaling pathways. Our results suggest an opposite regulation of these pathways by neurofibromin and the possible existence of a feedback loop sensing neurofibromin-related signaling. The present work illustrates the utility of PMOs to study alternative splicing that could be applied to other alternatively spliced genes in vitro and in vivo.

Development of antisense-mediated myostatin knockdown for the treatment of insulin resistance

Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes. However, there is currently no clinically effective myostatin inhibitor, and therefore novel methods are required. We evaluated the use of antisense phosphorodiamidate morpholino oligomers (PMO) to reduce myostatin expression in skeletal muscle and measured their effects on muscle mass and glucose uptake. C57/Bl6 mice received intramuscular or intravenous injections of anti-myostatin PMOs. Repeated intramuscular administration lead to a reduction in myostatin transcript levels (~ 20–40%), and an increase in muscle mass in chow and high-fat diet (HFD)-fed mice, but insulin-stimulated glucose uptake was reduced in PMO-treated muscles of HFD-fed mice. Five weekly intravenous administrations of 100 nmol PMO did not reduce myostatin expression, and therefore had no significant physiological effects. Unexpectedly, exon skipping levels were higher after intramuscular administration of PMO in HFD- than chow-fed mice. These results suggest that a modest PMO-induced reduction in myostatin transcript levels is sufficient to induce an increase in muscle mass, but that a greater degree of inhibition may be required to improve muscle glucose uptake.

The administration of antisense oligonucleotide golodirsen reduces pathological regeneration in patients with Duchenne muscular dystrophy

During the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.

Neutral Surfactant Enhanced Exon-skipping of Morpholino Oligonucleotides in vitro and in mdx Mice

Antisense oligomers induced exon-skipping has been promising therapy for Duchenne muscular dystrophy in preclinical and clinical trials, but its therapeutic potential could be improved with enhanced delivery approach. A few neutral surfactants were investigated here in for their performance to improve exon-skipping of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. This study showed that these surfactants, especially Bigchapand Deoxy Bigchap, improved the delivery efficiency of PMO to the levels comparable to Endoporter-mediated PMO delivery in vitro, and significant enhancement with Deoxy Bigchap-mediated PMO was further observed in mdx mice up to 7-fold compared with PMO alone. Cytotoxicity of the surfactants except for Mega10 was much lower than that by Endoporter in vitro and not clearly observed in vivo under the tested dosage. These results reveal that surfactant’s composition is key factor as delivery carrier to improve PMO exon-skipping efficiency, the efficacy and safety endow neutral surfactants as potential delivering enhancer for oligonucleotide in the treatment of muscular dystrophy or other diseases.

Xenopus gpx3 Mediates Posterior Development by Regulating Cell Death during Embryogenesis.

Glutathione peroxidase 3 (GPx3) belongs to the glutathione peroxidase family of selenoproteins and is a key antioxidant enzyme in multicellular organisms against oxidative damage. Downregulation of GPx3 affects tumor progression and metastasis and is associated with liver and heart disease. However, the physiological significance of GPx3 in vertebrate embryonic development remains poorly understood. The current study aimed to investigate the functional roles of gpx3 during embryogenesis. To this end, we determined gpx3’s spatiotemporal expression using Xenopus laevis as a model organism. Using reverse transcription polymerase chain reaction (RT-PCR), we demonstrated the zygotic nature of this gene. Interestingly, the expression of gpx3 enhanced during the tailbud stage of development, and whole mount in situ hybridization (WISH) analysis revealed gpx3 localization in prospective tail region of developing embryo. gpx3 knockdown using antisense morpholino oligonucleotides (MOs) resulted in short post-anal tails, and these malformed tails were significantly rescued by glutathione peroxidase mimic ebselen. The gene expression analysis indicated that gpx3 knockdown significantly altered the expression of genes associated with Wnt, Notch, and bone morphogenetic protein (BMP) signaling pathways involved in tailbud development. Moreover, RNA sequencing identified that gpx3 plays a role in regulation of cell death in the developing embryo. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone 3 (PH3) staining confirmed the association of gpx3 knockdown with increased cell death and decreased cell proliferation in tail region of developing embryos, establishing the involvement of gpx3 in tailbud development by regulating the cell death. Furthermore, these findings are inter-related with increased reactive oxygen species (ROS) levels in gpx3 knockdown embryos, as measured by using a redox-sensitive fluorescent probe HyPer. Taken together, our results suggest that gpx3 plays a critical role in posterior embryonic development by regulating cell death and proliferation during vertebrate embryogenesis.

Chemical Modulators of Fibrinogen Production and Their Impact on Venous Thrombosis

Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention.

A Zebrafish Model for Severe Congenital Neutropenia with Jagn1b-Deficiency

Severe congenital neutropenia (CN) is a bone marrow failure characterized by an impaired neutrophil development. This disease can be caused by different germline mutations, which one of them is Jagunal homolog 1 (JAGN1). CN patients are prone to develop life-threatening infections from birth on and the treatment of choice is recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, patients with JAGN1 deficiency poorly respond to this treatment. Therefore, the development of new therapeutic strategies to treat this disease is essential. Thus far, lack of an animal model was the main limitation to study the function of JAGN1 in neutropenia and perform high-throughput compound screening. In this work, we studied the role of jagn1b in zebrafish granulopoiesis and hematopoiesis. Two different approaches were used. First, we injected antisense morpholino that efficiently blocked the translation of jagn1b mRNA. As a second approach, we used the CRISPR-Cas9 technique to introduce mutations in the zebrafish jagn1b gene and analyzed the crispants. Both of these approaches were tested in wild-type embryos as well as the transgenic mpo:GFP reporter line, in which neutrophils are labeled with GFP. Our results showed that interference with Jagn1b reduced the number of neutrophils, without affecting the early myelopoiesis and monocytes/macrophages. Further analysis revealed that apoptosis was enhanced in embryos injected with the jagn1b morpholino, however, this effect was not restricted to the hematopoietic tissues. Our results also suggested that unfolded protein response (UPR) was activated because the expression levels of genes involved in this pathway such as chop, atf4a and atf4b were significantly upregulated in the jagn1b morphants, when compared with the wild-type counterparts. Overall, we have successfully established a novel in vivo model for studying the role of JAGN1 in granulopoiesis, and the possible role of Jagn1b in UPR response might be useful to find new targets for the development of therapeutic strategies for CN. Disclosures No relevant conflicts of interest to declare.

Differential gene expression of virulence factors modulates infectivity of TcI Trypanosoma cruzi strains.

Trypanosoma cruzi is the etiological agent of Chagas disease, whose clinical outcome ranges from asymptomatic individuals to chronic fatal megasyndromes. Despite being central to pathogenesis, the regulation of parasite virulence factors' expression remains largely unknown. In this work, the relative expression of several parasite virulence factors between two TcI strains (Ninoa, low virulence and Qro, high virulence) was assessed by qRT-PCR of total and of polysome-associated mRNA, as well as by western blots. Trypomastigotes were also incubated with specific anti-sense morpholino oligonucleotides to block the translation of a selected virulence factor, calreticulin, in both strains. Ninoa trypomastigotes showed significantly lower levels of trypomastigote-decay acceleration factor, complement regulatory protein, complement C2 receptor inhibitor trispanning, and glycoproteins 82 and 90 mRNAs compared with Qro. There was a significantly lower recruitment of complement regulatory protein and complement C2 receptor inhibitor trispanning mRNAs to polysomes and higher recruitment of MASP mRNA to monosomes in Ninoa strain. Calreticulin mRNA displayed both a higher total mRNA level and recruitment to translationally active polysomes in the Ninoa strain (low virulence) than in the Qro strain (high virulence). When calreticulin was downregulated by ≈ 50% by anti-sense morpholino oligonucleotides, a significant decrease of parasite invasion in mammalian cells was found in both strains. Calreticulin downregulation, however, only increased significantly the activation of the complement system by Ninoa trypomastigotes. These results suggest a role for the regulation of virulence factors' gene expression in the differential virulence among T. cruzi strains. Furthermore, a possible function of calreticulin in parasite invasion not related to its binding to complement factors is shown.

Internal Oligoguanidinium Transporter: Mercury-Free Scalable Synthesis, Improvement of Cellular Localization, Endosomal Escape, Mitochondrial Localization, and Conjugation with Antisense Morpholino for NANOG Inhibition to Induce Chemosensitization of Taxol in MCF-7 Cells.

A nontoxic delivery vehicle is essential for the therapeutic applications of antisense phosphorodiamidate morpholino oligonucleotides (PMOs). Though guanidinium-rich or arginine-rich cellular transporter conjugated Vivo-PMO or PPMO has been developed for in vivo application, however, either their toxicity or stability has become an issue. Previously, we reported nonpeptidic internal guanidinium transporter (IGT) mediated delivery of PMO for gene silencing and got encouraging results. In this paper, we report the synthesis of IGT using a Hg-free method for scale up and N-terminal modification of IGT with a suitable hydrophobic or lipophilic group to improve the cell permeability, endosomal escape, and mitochondrial localization and to reduce toxicity in the MTT assay. For the delivery of PMO, IGT-PMO conjugate was synthesized to target NANOG in cells, a transcription factor required for cancer stem cell proliferation and embryonic development and is involved in many cancers. Our data shows IGT-PMO-facilitated NANOG inhibition, and thereby the prevention of EpCAM-N-Cadherin-Vimentin axis mediated epithelial to mesenchymal transition (EMT) in MCF-7 cells. Moreover, unlike taxol, NANOG inhibition influences the expression of stemness factor c-Myc, Hh-Gli signaling proteins, other cancer related factors, and their respective phenotypes in cancer cells. To the best of our knowledge, this is the first report to illustrate that the IGT-PMO-mediated NANOG inhibition increases the therapeutic potential of taxol and induces G0-G1 arrest in cancer cells to prevent cancer progression. However, it warrants further investigation in other cancer cells and preclinical platforms.

The low affinity p75 neurotrophin receptor is down-regulated in congenital anomalies of the kidney and the urinary tract: Possible involvement in early nephrogenesis

Congenital Anomalies of the Kidney and of the Urinary Tract (CAKUT) cover a broad range of disorders including abnormal kidney development caused by defective nephrogenesis. Here we explored the possible involvement of the low affinity p75 neurotrophin receptor (p75NTR) in CAKUT and nephrogenesis. In mouse, p75NTR was highly expressed in fetal kidney, located within cortical early nephrogenic bodies, and decreased rapidly after birth. In human control fetal kidney, p75NTR was also located within the early nephrogenic bodies as well as in the mature glomeruli, presumably in the mesangium. In CAKUT fetal kidneys, the kidney cortical structure and the localization of p75NTR were often disorganized, and quantification of p75NTR in amniotic fluid revealed a significant reduction in CAKUT compared to control. Finally, invalidation of p75NTR in zebrafish embryo with an antisense morpholino significantly altered pronephros development. Our results indicate that renal p75NTR is altered in CAKUT fetuses, and could participate to early nephrogenesis.

Enhanced Canonical Wnt Signaling During Early Zebrafish Development Perturbs the Interaction of Cardiac Mesoderm and Pharyngeal Endoderm and Causes Thyroid Specification Defects.

Background: Congenital hypothyroidism due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the majority of cases. This situation reflects, in part, our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification. Methods: We treated zebrafish embryos during different developmental periods with small-molecule compounds known to manipulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. We used the antisense morpholino (MO) technique to create a zebrafish acardiac model. For thyroid rescue experiments, bone morphogenetic protein (BMP) pathway induction in zebrafish embryos was obtained by manipulation of heat-shock inducible transgenic lines. Results: Combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of MO-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development. Conclusion: Collectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.