Antisense Morpholino Oligonucleotides Research Articles

Synthesis of Nucleobase-Functionalized Morpholino Monomers.

Morpholino antisense oligonucleotides are used as routine tools in developmental biology to investigate gene function during early embryogenesis. These chemically modified oligos contain morpholine ring connected with phosphorodiamidate linkages as backbone but carry unmodified nucleobases. In this chapter, we describe the methods to further modify the nucleobases using palladium-catalyzed cross-coupling reactions. The key reactions used are halogenations of the nucleobases in suitable position and subsequent Pd-catalyzed Sonogashira and Suzuki reactions. The sequential synthetic steps are described in detail in this chapter, and the examples are shown in tables.

Methods of Calpain Inhibition to Determine the Role of Calpains in Embryo Development in Amphibians.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO). We also provide methods to determine the effectiveness of the calpain inhibition and effect on cell fate specification and morphogenetic movements, during embryogenesis in vivo.

U1 snRNP Telescripting Roles in Transcription and Its Mechanism.

Telescripting is a fundamental cotranscriptional gene regulation process that relies on U1 snRNP (U1) to suppress premature 3'-end cleavage and polyadenylation (PCPA) in RNA polymerase II (Pol II) transcripts, which is necessary for full-length transcription of thousands of protein-coding (pre-mRNAs) and long noncoding (lncRNA) genes. Like U1 role in splicing, telescripting requires U1 snRNA base-pairing with nascent transcripts. Inhibition of U1 base-pairing with U1 snRNA antisense morpholino oligonucleotide (U1 AMO) mimics widespread PCPA from cryptic polyadenylation signals (PASs) in human tissues, including PCPA in introns and last exons' 3'-untranslated regions (3' UTRs). U1 telescripting-PCPA balance changes generate diverse RNAs depending on where in a gene it occurs. Long genes are highly U1-telescripting-dependent because of PASs in introns compared to short genes. Enrichment of cell cycle control, differentiation, and developmental functions in long genes, compared to housekeeping and acute cell stress response genes in short genes, reveals a gene size-function relationship in mammalian genomes. This polarization increased in metazoan evolution by previously unexplained intron expansion, suggesting that U1 telescripting could shift global gene expression priorities. We show that that modulating U1 availability can profoundly alter cell phenotype, such as cancer cell migration and invasion, underscoring the critical role of U1 homeostasis and suggesting it as a potential target for therapies. We describe a complex of U1 with cleavage and polyadenylation factors that silences PASs in introns and 3' UTR, which gives insights into U1 telescripting mechanism and transcription elongation regulation.

Microinjection methods for sea urchin eggs and blastomeres.

Methods for microinjection into sea urchin eggs have become relatively easier because of the technical improvements by a number of researchers in the past decades. However, the size and the characteristics, such as the elasticity and toughness, of the eggs and embryos differ in species, so that we still need to modify the details of methods to adapt to each target. In this section, I list microinjection methods for three species: Hemicentrotus pulcherrimus, which has relatively tough eggs, Temnopleurus reevesii, which has slightly weak eggs, and Strongylocentrotus purpuratus, which is the most used species in sea urchin biology. In addition, I describe the methods for co-injection of morpholino anti-sense oligonucleotides and mRNAs, as well as the method for microinjection into blastomeres.

Lysine demethylase 3a in craniofacial and neural development during Xenopus embryogenesis.

Epigenetic modifier lysine demethylase 3a(Kdm3a) specifically demethylates mono‑ and di‑methylated ninth lysine of histone3 and belongs to the Jumonji domain‑containing group of demethylases. Kdm3a serves roles during various biological and pathophysiological processes, including spermatogenesis and metabolism, determination of sex, androgen receptor‑mediated transcription and embryonic carcinoma cell differentiation. In the present study, physiological functions of Kdm3a were evaluated during embryogenesis of Xenopus laevis. Spatiotemporal expression pattern indicated that kdm3a exhibited its expression from early embryonic stages until tadpole stage, however considerable increase of kdm3a expression was observed during the neurula stage of Xenopus development. Depleting kdm3a using kdm3a antisense morpholino oligonucleotides induced anomalies, including head deformities, small‑sized eyes and abnormal pigmentation. Whole‑mount insitu hybridization results demonstrated that kdm3a knockdown was associated with defects in neural crest migration. Further, quantitative polymerase chain reaction revealed abnormal expression of neural markers in kdm3a morphants. RNA sequencing of kdm3a morphants indicated that kdm3a was implicated in mesoderm formation, cell adhesion and metabolic processes of embryonic development. In conclusion, the results of the present study indicated that Kdm3a may serve a role in neural development during Xenopus embryogenesis and may be targeted for treatment of developmental disorders. Further investigation is required to elucidate the molecular mechanism underlying the regulation of neural development by Kdm3a.

Physiological effects of KDM5C on neural crest migration and eye formation during vertebrate development

BackgroundLysine-specific histone demethylase 5C (KDM5C) belongs to the jumonji family of demethylases and is specific for the di- and tri-demethylation of lysine 4 residues on histone 3 (H3K4 me2/3). KDM5C is expressed in the brain and skeletal muscles of humans and is associated with various biologically significant processes. KDM5C is known to be associated with X-linked mental retardation and is also involved in the development of cancer. However, the developmental significance of KDM5C has not been explored yet. In the present study, we investigated the physiological roles of KDM5C during Xenopus laevis embryonic development.ResultsLoss-of-function analysis using kdm5c antisense morpholino oligonucleotides indicated that kdm5c knockdown led to small-sized heads, reduced cartilage size, and malformed eyes (i.e., small-sized and deformed eyes). Molecular analyses of KDM5C functional roles using whole-mount in situ hybridization, β-galactosidase staining, and reverse transcription-polymerase chain reaction revealed that loss of kdm5c resulted in reduced expression levels of neural crest specifiers and genes involved in eye development. Furthermore, transcriptome analysis indicated the significance of KDM5C in morphogenesis and organogenesis.ConclusionOur findings indicated that KDM5C is associated with embryonic development and provided additional information regarding the complex and dynamic gene network that regulates neural crest formation and eye development. This study emphasizes the functional significance of KDM5C in Xenopus embryogenesis; however, further analysis is needed to explore the interactions of KDM5C with specific developmental genes.

Mutation-Specific Dose Reduction in Functional SRP54 Protein Causes Isolated or Syndromic Neutropenia with Shwachman-Diamond-like Features

Sequencing analyses are increasingly performed on patients presenting with suspected congenital disease but lacking classical mutations linked to the presented phenotype. However, in case a novel mutation is found, its causal contribution to the patients' clinical symptoms is yet unclear and requires further exploration in functional studies. Here we use the zebrafish model to analyze the functional relevance of heterozygous SRP54 gene mutations identified in patients with unexplained neutropenia (T117del) or Shwachman-Diamond like disease involving severe neutropenia and exocrine pancreas insufficiency (T115A, G226E) (Carapito et al, 2017). The function of wildtype SRP54 protein was explored in hematopoiesis and pancreas development using two different antisense morpholino oligonucleotides (MO) and a zebrafish srp54 mutant. Reduced neutrophil numbers were observed in MO versus control injected fish when analyzed by WISH for mpx or using Tg(lyz:DsRed) and Tg(mpx:eGFP) lines. Morphants displayed not only quantitatively but also qualitatively impaired neutrophils, which migrated less to injury sites in tail fin injury assays. Furthermore, exocrine but not endocrine pancreas impairment was observed in MO versus control injected fish. Both neutrophiles and exocrine pancreas development could be rescued by co-injection with wildtype SRP54 mRNA.Interestingly, in the corresponding Sanger mutant, homozygous srp54-/- fish display not only most severe neutropenia but also gross developmental defects which cause early lethality at 2 days post-fertilization and thus impede assessment of pancreas development. Heterozygous srp54+/- fish are viable, show less severe neutropenia than homozygous fish and, interstingly, have no exocrine pancreas defect. The delineated experiments explore thus the effects of different dosages of residual functional SRP54 protein (wildtype > srp54+/- > morpholino treated > srp54-/-) on phenotypic manifestation. The obtained results indicate that the dose of residual SRP54 protein that can functionally act within the SRP may dictate disease phenotype in patients with SRP54 mutations (neutropenia only > more severe neutropenia and exocrine pancreas defects > most severe neutropenia, early lethality).We thus hypothesize that the different mutations observed in patients differentially affect the functionality of the residual wildtype SRP54 protein and thus associate with isolated neutropenia (T117del) or more severe disease with Shwachman-Diamond like features (T115A, G226E). To mimic the heterozygote mutation status observed in patients, we injected the different mutated mRNAs in srp54+/- zebrafish. None of the injected mRNAs was able to rescue the neutropenia phenotype, indicating that all mutated proteins are functionally impaired. Confirming our hypothesis, injection of T115A and G226E in fact further aggravated neutropenia and additionally induced exocrine pancreas defects, while T117del did not.Taken together, these analyses support the notion that full deletion of SRP54 is not compatible with life and therefore homozygous SRP54 are not observed in patients. Instead, heterozygous SRP54 mutations may cause only neutropenia or alternatively the more severe Shwachman-Diamond like syndrom, depending on the nature of the underlying mutation (either functional null or dominant negative). DisclosuresNo relevant conflicts of interest to declare.

CPSF1-Mediated Acute Myeloid Leukemia Fusion 3'UTR Alternation Is a Potential Therapeutic Target

Alternative polyadenylation (APA) can alter the three prime untranslated region (3'UTR) length of mRNAs, crucial for regulating mRNA metabolism and gene expression. Despite the prevalence of APA post-transcriptional regulation in cancers, changes in 3'UTR length by APA and its contribution to leukemia development have not been thoroughly studied. In this study, we demonstrated the significance of APA of leukemic fusion genes in acute myelogenous leukemia (AML) development.T(8;21) is the most common chromosomal abnormality in AML and encodes the AML1-ETO (AE) fusion gene. The AE 3'UTR has a full length of 5.2kb that contains 4 canonical polyadenylation sites (PAS), such that APA can result in several mRNA isoforms with varying 3'UTR lengths. However, RNA-seq and absolute quantification qPCR revealed that AE mainly uses 2 PAS: 5.2kb (long 3'UTR) and 3.7kb (short 3'UTR). Moreover, the AE short 3'UTR is the major isoform in both t(8;21) AML primary patients and cell lines. Based on these findings, we next hypothesize that changes in PAS usage and thus AE 3'UTR length, can modulate fusion gene expression.The single cell based dynamic array revealed that AE+ non-leukemic differentiated cells coexisted with AML blasts in the diagnostic bone marrow and these non-leukemic AE+ cells expressed much lower AE compared to AML blasts (monocytes, 0.19-fold and granulocytes, 0.11-fold, p<0.001). Among these leukemic and AE+ non-leukemic cells, AE expression and long 3'UTR usage showed a significant negative correlation (R2=0.64, p<0.01). Moreover, half-life assay using actinomycin D showed that AE long 3'UTR had much shorter half-life than AE short 3'UTR (0.8 hour and 2.8 hours, p<0.01). A dual reporter assay also presented the predominant repression of AE long 3'UTR (3.7 kb: 0.31 and 5.2 kb: 0.08, p<0.001). These data suggest that AE 3'UTR length affects mRNA stability and is an important indicator of fusion gene expression.The selective inhibition of AE short 3'UTR PAS by morpholino antisense oligonucleotide caused an increase of AE long 3'UTR usage, decrease of total AE expression, and an increase in apoptosis. These results indicate that the inhibition of AE short 3'UTR PAS increases the long 3'UTR usage and the PAS usage of leukemic fusion genes could be a potential therapeutic target.To find the key regulator of this 3'UTR change, we comprehensively knocked down all APA machinery members by shRNA in t(8;21) AML cell lines. We discovered that CPSF1 (cleavage and polyadenylation specific factor 1) knockdown increased long 3'UTR usage (37% in total AE) and down-regulated overall AE mRNA (0.47-fold). Importantly, CPSF1 was highly expressed in t(8;21) AML patients' leukemia cells compared to healthy CD34+ cells (2.7-fold, p=0.01). CPSF1 knockdown also promoted apoptosis and Gene Set Enrichment Analysis revealed that CPSF1 knockdown showed the similar gene signature to AE knockdown. These data suggest that CPSF1 promotes AE 3'UTR shortening, resulting in AE stability and a growth advantage of AML cells. Importantly, we also found that AE regulates CPSF1 expression, suggesting a positive feedback loop of AML1-ETO and CPSF1 in t(8;21) AML.Finally, we also checked APA of leukemic fusion gene MLL-AF9, which has 2 canonical PAS (0.1kb short 3'UTR and 0.7kb long 3'UTR). Compared to healthy CD34+ cells, THP-1 cells (MLL-AF9+ AML cell line) predominantly used MLL-AF9 short 3'UTR. Knockdown of CPSF1, which is also up-regulated in MLL-associated AML, showed increase of MLL-AF9 long 3'UTR usage, decrease of MLL-AF9 expression, and an increase of apoptosis in THP-1 cells.Collectively, we conclude that the polyadenylation regulator CPSF1 controls 3'UTR length of leukemic fusion transcripts, and extending the fusion gene 3'UTR by knocking down CPSF1 reduces fusion gene mRNA stability and restricts cell growth in both AE+ and MLL-AF9+ AMLs. These results suggest that 3'UTR shortening of oncogenic fusion gene transcripts contributes to leukemogenesis and controlling CPSF1 and 3'UTR usage may be a useful approach to inhibit leukemia progression. DisclosuresAkashi:Pfizer: Research Funding; Taiho Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Eli Lilly Japan: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Research Funding; MSD: Research Funding; sanofi: Research Funding; Novartis pharma: Research Funding; Chugai Pharma: Research Funding; Celgene: Research Funding, Speakers Bureau; Asahi-kasei: Research Funding; Ono Pharmaceutical: Research Funding.

Ing4 Regulates Hematopoiesis through Suppression of NF-Kb

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In the absence of Ing4, there is a significant increase in NF-kB target gene expression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis, where Ing4 deficiency impairs hematopoietic stem cell (HSC) function, but enhances multipotent progenitor cell (MPP) regenerative capacity. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance.To define the role of Ing4 in zebrafish HSPCs, we designed an anti-sense morpholino oligo against Ing4 and injected into zebrafish embryos at the single cell stage. Embryos were screened using in situ hybridizations for c-myb and runx1 expression, which are highly expressed in the aorta, gonad, mesonephros (AGM) region in the developing zebrafish embryo. We found that Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the AGM, demonstrating a lack of HSPC specification. Analysis of ephrinB2 expression showed normal specification of the aorta in Ing4 morphant embryos, signifying that the step of HSPC specification is affected in the absence of Ing4. Overexpression of human Ing4 in zebrafish embryos resulted in increased HSPC marker staining suggesting that normal expression levels of Ing4 are required for HSC specification.As Ing4 is an epigenetic regulator that binds specific gene loci, we examined the chromatin occupancy of Ing4 in human peripheral blood CD34+ progenitor cells. Using ChIP-seq for Ing4 in CD34+ cells, we show that Ing4 binds to many regulators of blood development including MYB, LMO2, RUNX1, and IKAROS, and several NF-kB target genes. In other tissues, Ing4 negatively regulates NF-kB, so accordingly, loss of Ing4 results in an overabundance of NF-kB signaling. To address NF-kB target gene expression in Ing4-deficient zebrafish embryos, we performed qPCR analysis at 36hpf. These assays showed an increase in the expression of a subset of NF-kB target genes (IKBKE, IL-19, IL-1b, IL-20R). Simultaneous knockdown of both Ing4 and RelA, through combined morpholino injections against both factors, resulted in the rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the loss of Ing4.A mouse model for Ing4 deficiency was generated to further evaluate the role of Ing4 in differentiated immune cells. These mice are developmentally normal but are hypersensitive to stimulation with LPS. Interestingly, we found that Ing4-/- mice showed skewed hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. Based on these exciting findings, we hypothesize that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. DisclosuresNo relevant conflicts of interest to declare.

Radiation Enhances the Delivery of Antisense Morphalino Oligonucleotides across the Blood-Brain Barrier

We previously demonstrated that non-ablative ionizing radiation enhanced the delivery of intravenously administered unmodified antisense morpholino oligonucleotides (aONs) to subcutaneous tumor sites and results in 50% reduction in the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT), compared to non-radiated subcutaneous tumor sites in the same animal. Delivery of aONs across the blood-brain barrier (BBB) remains a major challenge. Ionizing radiation is well known to alter BBB permeability. We hypothesized that ionizing radiation will facilitate the delivery of IV aONs across the BBB. Long Evans rats received either 5 Gy whole brain radiation, or no radiation (n=6 per group); 24 h later, half of each group received GFP-tagged nonspecific unmodified morpholino aON (10.5 mg/kg IV). Brains were harvested after perfusion with saline 24h after aON administration. GFP localization was assessed in 50 μm sections using a laser-scanning confocal microscope. We observed increased delivery of the morpholino aONs into the brain parenchyma only in the pre-radiated cohort. Rats that received radiation followed by IV GFP-conjugated aONs showed a punctate green fluorescent signal intracellularly in perivascular cells. The punctate staining pattern detected is suggestive that radiation enhanced the endocytosis of aONs and uptake into endosomes and lysosomes. Radiation enhances the targeted delivery of unmodified aONs across the BBB and uptake by perivascular cells. This observation is logical as radiation is known to enhance cellular endocytosis. Morpholino aONs are stable at physiologic pH and can be tailored to silence many key cancer proteins. This approach holds significant translational potential since radiation is already an integral part of brain tumor therapies. Follow-up studies will investigate if radiation enhances the targeted delivery of aONs in a rodent MGMT expressing-brain tumor model to inhibit MGMT expression and function as a chemo-radiation sensitizer.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

TBX20 Regulates Angiogenesis Through the Prokineticin 2-Prokineticin Receptor 1 Pathway.

Angiogenesis is integral for embryogenesis, and targeting angiogenesis improves the outcome of many pathological conditions in patients. TBX20 is a crucial transcription factor for embryonic development, and its deficiency is associated with congenital heart disease. However, the role of TBX20 in angiogenesis has not been described. Loss- and gain-of-function approaches were used to explore the role of TBX20 in angiogenesis both in vitro and in vivo. Angiogenesis gene array was used to identify key downstream targets of TBX20. Unbiased gene array survey showed that TBX20 knockdown profoundly reduced angiogenesis-associated PROK2 (prokineticin 2) gene expression. Indeed, loss of TBX20 hindered endothelial cell migration and in vitro angiogenesis. In a murine angiogenesis model using subcutaneously implanted Matrigel plugs, we observed that TBX20 deficiency markedly reduced PROK2 expression and restricted intraplug angiogenesis. Furthermore, recombinant PROK2 administration enhanced angiogenesis and blood flow recovery in murine hind-limb ischemia. In zebrafish, transient knockdown of tbx20 by morpholino antisense oligos or genetic disruption of tbx20 by CRISPR/Cas9 impaired angiogenesis. Furthermore, loss of prok2 or its cognate receptor prokr1a also limited angiogenesis. In contrast, overexpression of prok2 or prokr1a rescued the impaired angiogenesis in tbx20-deficient animals. Our study identifies TBX20 as a novel transcription factor regulating angiogenesis through the PROK2-PROKR1 (prokineticin receptor 1) pathway in both development and disease and reveals a novel mode of angiogenic regulation whereby the TBX20-PROK2-PROKR1 signaling cascade may act as a "biological capacitor" to relay and sustain the proangiogenic effect of vascular endothelial growth factor. This pathway may be a therapeutic target in the treatment of diseases with dysregulated angiogenesis.

Polarity and morphogenesis of the eye epithelium requires the adhesion junction associated adaptor protein Traf4

ABSTRACTDuring development, neuroepithelial progenitors acquire apico-basal polarity and adhere to one another via apically located tight and adherens junction complexes. This polarized neuroepithelium must continue to integrate cells arising through cell divisions and intercalation, and allow for cell movements, at the same time as undergoing morphogenesis. Cell proliferation, migration and intercalation all occur in the morphing embryonic eye. To understand how eye development might depend on dynamic epithelial adhesion, we investigated the function of a known regulator of junctional plasticity, Tumour necrosis factor receptor-associated factor 4 (Traf4). traf4a mRNA is expressed in the developing eye vesicle over the period of optic cup morphogenesis, and Traf4a loss leads to disrupted evagination and elongation of the eye vesicles, and aberrant organization and apico-basal polarity of the eye epithelium. We propose a model whereby Traf4a regulates apical junction plasticity in nascent eye epithelium, allowing for its polarization and morphogenesis.Symbols and Abbreviations: AB: apico-basal; aPKC: atypical protein kinase-C; CRISPR: clustered regularly-interspaced short palindromic repeats; GFP: green fluorescent protein; hpf: hours post-fertilization; MO: antisense morpholino oligonucleotide; pHH3: phospho histone H3; ss: somite stage; Traf4: Tumour necrosis factor receptor-associated factor 4; ZO-1: zona occludens-1

Blocking the 5\u2032 splice site of exon 4 by a morpholino oligomer triggers APOL1 protein isoform switch

APOL1 risk alleles G1 or G2 are associated with a kidney disease phenotype exclusively in people of recent African ancestry. Here we show that exon 4 encoding a part of the APOL1 signal peptide is constitutively spliced in major APOL1 transcripts expressed in kidney glomerular and tubular cells. We demonstrate that constitutive splicing of exon 4 results from a suboptimal hnRNP A1 binding motif found in exon 4. Accordingly, a robust binding of hnRNP A1 protein to a consensus hnRNP A1 cis-acting element in exon 4 results in almost complete exclusion of exon 4 from the APOL1 minigene transcripts. Blocking the 5′ splice site at the exon 4/intron boundary with a specific antisense morpholino oligonucleotide excludes exon 4 from the splicing pattern of endogenous APOL1 transcripts. These transcripts are fully functional and produce APOL1 protein isoform that is not normally detectable in podocytes. Together with our previous data showing no cytotoxicity of overexpressed APOL1 isoform lacking exon 4, we propose that morpholino-induced APOL1 isoform switch may provide a new tool to identify in vivo molecular mechanism(s) by which risk alleles promote or mediate the kidney disease phenotype.

The N-terminal domain of gastrulation brain homeobox 2 (Gbx2) is required for iridophore specification in zebrafish

Although body color pattern formation by pigment cells plays critical roles in animals, pigment cell specification has not yet been fully elucidated. In zebrafish, there are three chromatophores: melanophore, iridophore, and xanthophore, that are derived from neural crest cells (NCCs). A recent study has reported the differentially expressed genes between melanophores and iridophores. Based on transcriptome data, we identified that Gbx2 is required for iridophore specification during development. In support of this, iridophore formation is suppressed by gbx2 knockdown by morpholino antisense oligonucleotide, at 72 h post fertilization (hpf) in zebrafish. Moreover, gbx2 is expressed in sox10-expressing NCCs and guanine crystal plates-containing iridophores during development at 24 and 48 hpf, respectively. In gbx2 knockdown zebrafish embryos, apoptosis of sox10-expressing NCCs was detected at 24 hpf without any effect on the formation of melanophores and xanthophores at 48 hpf. We further observed that the N-terminal domain of Gbx2 is able to rescue the iridophore formation defect caused by gbx2 knockdown. Our study provides insights into the requirement of N-terminal domain of Gbx2 for iridophore specification in zebrafish.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord.

Microinjection of mRNAs and Oligonucleotides.

Microinjecting lineage tracers into a single blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified blastomere to target altered gene expression specifically to its lineage. Xenopus blastomeres are robust and tolerate pressure-driven microinjection up to a few hundred cells, and they efficiently translate exogenously supplied mRNAs. Targeted microinjections, described here, significantly reduce off-target effects of the mRNAs or oligonucleotides. Many types of constructs can be synthesized to provide specific information about gene function. For example, microinjecting mRNA encoding the wild-type gene in its normal expression domain or in an ectopic site tests whether it promotes or represses target genes or alters the formation of tissues of interest. Mutant forms of a gene transcript can illuminate the function of different domains of the encoded protein or show the developmental consequences of a mutation found in a human disease. mRNAs encoding dominant-negative forms of a protein can elicit a functional knockdown and thereby establish the necessity for that gene in a developmental process. Microinjecting antisense morpholino oligonucleotides (MOs) that are designed to block either endogenous mRNA translation or splicing is an effective method to reduce the levels of endogenous protein.

Methods of In Vivo Gene-Specific Knockdown Using Morpholino and Vivo-Morpholino Oligonucleotides

The functioning of the small GTPase gene, Ras-dva1, has been studied during regeneration processes of the tadpole tails of the clawed frog Xenopus laevis. For this purpose, we have developed two approaches for the gene knockdown using injections of sequence-specific antisense morpholino oligonucleotides (MO) or vivo-morpholino oligonucleotides (vivo-MO). It has been shown for the first time that intracellular Ras-dva-specific MO injected into Xenopus 4–16 of blastomere embryos or vivo-MO injected directly into the distal part of the tadpole tail at stages 40–41 efficiently inhibit the Ras-dva1 gene functioning and leads to the same morphological malformations of the tail regeneration. Using qRT-PCR, we detected the inhibition of expression of the regeneration marker genes (Fgf20a and Msx1b) on the first day after amputation in the tail tissues of tadpoles with the Ras-dva1 knockdown.

Calcium signals act through histone deacetylase to mediate pronephric kidney morphogenesis.

Autosomal dominant polycystic kidney disease is the most common monogenetic kidney disorder and is linked to mutations in PKD1 and PKD2. PKD2, a Ca2+ -conducting TRP channel enriched in ciliated cells and gated by extracellular signals, is necessary to activate the multifunctional Ca2+/ calmodulin-dependent protein kinase type 2 (CaMK-II), enabling kidney morphogenesis and cilia stability. In this study, antisense morpholino oligonucleotides and pharmacological compounds were employed to investigate the roles of class II HDAC family members (HDAC 4, 5, and 6) in Zebrafish kidney development. While all three class II HDAC genes were expressed throughout the embryo during early development, HDAC5-morphant embryos exhibited anterior cysts and destabilized cloacal cilia, similar to PKD2 and CaMK-II morphants. In contrast, HDAC4-morphant embryos exhibited elongated cloacal cilia and lacked anterior kidney defects. Suppression of HDAC4 partially reversed the cilia shortening and anterior convolution defects caused by CaMK-II deficiency, whereas HDAC5 loss exacerbated these defects. EGFP-HDAC4, but not EGFP-HDAC5, translocated into the nucleus upon CaMK-II suppression in pronephric kidney cells. These results support a model by which activated CaMK-II sequesters HDAC4 in the cytosol to enable primary cilia formation and kidney morphogenesis. Developmental Dynamics 247:807-817, 2018. © 2018 Wiley Periodicals, Inc.

Roles of progesterone receptor membrane component 1 and membrane progestin receptor alpha in regulation of zebrafish oocyte maturation

Although previous studies suggest membrane progesterone receptor alpha (mPRα/Paqr7) mediates 17, 20β-dihydroxy-4-pregnen-3-one (DHP) induction of oocyte maturation (OM) in zebrafish, critical information needed to establish mPRα as the receptor mediating OM is lacking. The relative potencies of progestins and specific mPRα agonists in inducing OM matched their relative binding affinities for zebrafish mPRα, supporting its role in OM. Microinjection of pertussis toxin blocked DHP induction of OM and the progestin-induced decrease in cyclic AMP levels, suggesting mPRα activates an inhibitory G protein (Gi). Microinjection of morpholino antisense oligonucleotides to zebrafish pgrmc1 blocked induction of OM by DHP which was accompanied by decreased levels of Pgrmc1 and mPRα on the oocyte plasma membranes. Similarly, treatment of denuded oocytes with a PGRMC1 inhibitor, AG205, blocked the gonadotropin-induced increase in plasma membrane mPRα levels and attenuated DHP induction of OM. Co-incubation with two inhibitors of epidermal growth factor Erbb2, ErbB2 inhibitor II and AG 879, prevented induction of OM by DHP, indicating the likely involvement of Erbb2 in mPRα-mediated signaling. Treatment with AG205 reversed the inhibitory effects of the Erbb2 inhibitors on OM and also inhibited insulin-like growth factor-1 induction of OM. Close associations between Pgrmc1 and mPRα, and between Pgrmc1 and Erbb2 were detected in zebrafish oocytes with in situ proximity ligation assays. The results suggest progestin induction of OM in zebrafish is mediated through an mPRα/Gi/Erbb2 signaling pathway that requires Pgrmc1 for expression of mPRα on oocyte membranes and that Pgrmc1 also is required for induction of OM through Erbb2.