scholarly journals Identification of maternal-effect genes in zebrafish using maternal crispants

Development ◽  
2021 ◽  
Author(s):  
Cara E. Moravec ◽  
Gabriella C. Voit ◽  
Jarred Otterlee ◽  
Francisco Pelegri
Keyword(s):  
Maternal Effect ◽  
Reverse Genetics ◽  
Germ Line ◽  
Maternal Factors ◽  
Single Generation ◽  
Maternal Effect Genes ◽  
Biallelic Mutations ◽  
Genome Activation

In animals, early development is dependent on a pool of maternal factors, both RNA and proteins, which are required for basic cellular process and cell differentiation until zygotic genome activation. The role of a majority of these maternally expressed factors is not fully understood. By exploiting the biallelic editing ability of CRISPR-Cas9, we identify and characterize maternal-effect genes in a single generation, using a maternal crispant technique. We validated the ability to generate biallelic mutations in the germline by creating maternal crispants that phenocopied previously characterized maternal-effect genes: motley/birc5b, tmi/prc1l, and aura/mid1ip1. Additionally, by targeting maternally expressed genes of unknown function in zebrafish, we identified two new maternal-effect zebrafish genes, kpna7 and fhdc3. The genetic identity of these maternal crispants was confirmed by sequencing haploid progeny from F0 females, which allowed the analysis of newly induced lesions in the maternal germ line. Our studies show that maternal crispants allow for the effective identification and primary characterization of maternal-effect genes in a single generation, facilitating the reverse genetics analysis of maternal factors that drive embryonic development.

scholarly journals Identification of Maternal-Effect Genes in Zebrafish using Maternal Crispants

2021 ◽  
Author(s):  
Cara E. Moravec ◽  
Gabriella C. Voit ◽  
Jarred Otterlee ◽  
Francisco Pelegri
Keyword(s):  
Early Development ◽  
Maternal Effect ◽  
Reverse Genetics ◽  
Germ Line ◽  
Maternal Factors ◽  
Zebrafish Model ◽  
Single Generation ◽  
Maternal Effect Genes ◽  
Biallelic Mutations ◽  
Genome Activation

AbstractIn animals, early development is dependent on a pool of maternal factors, both RNA and proteins, which are required for basic cellular process and cell differentiation until zygotic genome activation. The role of a majority of these maternally expressed factors in adult fertility and early development is not fully understood. By exploiting the biallelic editing ability of CRISPR-Cas9 and the benefits of the zebrafish model, we identify and characterize maternal-effect genes in a single generation, using a maternal crispant technique. We validated the ability to generate biallelic mutations in the germline by creating maternal crispants that phenocopied previously characterized maternal-effect genes: motley/birc5b, tmi/prc1l, and aura/mid1ip1. Additionally, by targeting maternally expressed genes of unknown function in zebrafish, we identified two new maternal-effect zebrafish genes, kpna7 and fhcd3. The genetic identity of these maternal crispants was confirmed by sequencing haploid progeny from F0 females, which allowed the sequence analysis of newly induced lesions in the maternal germ line. Analysis of the induced lesions shows minimal genetic variation within a clutch, with an average of two edited alleles per clutch. These findings are consistent with biallelic editing events occurring in germ cells or their precursors of early CRISPR-Cas9-injected embryos, leading to maternal-effect phenotypes in the offspring. Our studies show that maternal crispants allow for the effective identification and primary characterization of maternal-effect genes in a single generation, facilitating the reverse genetics analysis of maternal factors that drive embryonic development.

scholarly journals Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

Biology ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 102
Author(s):  
De-Li Shi
Keyword(s):  
Germ Line ◽  
Gene Products ◽  
Transgenic Expression ◽  
Guide Rna ◽  
Guide Rnas ◽  
Transgenic Embryos ◽  
Mutant Phenotypes ◽  
Biallelic Mutations ◽  
Genome Activation ◽  
Maternal Gene

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

scholarly journals The miR-125 family is an important regulator of the expression and maintenance of maternal effect genes during preimplantational embryo development

Open Biology ◽  
2016 ◽  
Vol 6 (11) ◽  
pp. 160181 ◽  
Author(s):  
Kyeoung-Hwa Kim ◽  
You-Mi Seo ◽  
Eun-Young Kim ◽  
Su-Yeon Lee ◽  
Jini Kwon ◽  
...  
Keyword(s):  
Embryo Development ◽  
Maternal Effect ◽  
Early Embryo ◽  
Binding Motif ◽  
Seed Region ◽  
Maternal Effect Gene ◽  
Maternal Effect Genes ◽  
Important Regulator ◽  
Genome Activation ◽  
Zygotic Genome

Previously, we reported that Sebox is a new maternal effect gene (MEG) that is required for early embryo development beyond the two-cell (2C) stage because this gene orchestrates the expression of important genes for zygotic genome activation (ZGA). However, regulators of Sebox expression remain unknown. Therefore, the objectives of the present study were to use bioinformatics tools to identify such regulatory microRNAs (miRNAs) and to determine the effects of the identified miRNAs on Sebox expression. Using computational algorithms, we identified a motif within the 3′UTR of Sebox mRNA that is specific to the seed region of the miR-125 family, which includes miR-125a-5p, miR-125b-5p and miR-351-5p. During our search for miRNAs, we found that the Lin28a 3′UTR also contains the same binding motif for the seed region of the miR-125 family. In addition, we confirmed that Lin28a also plays a role as a MEG and affects ZGA at the 2C stage, without affecting oocyte maturation or fertilization. Thus, we provide the first report indicating that the miR-125 family plays a crucial role in regulating MEGs related to the 2C block and in regulating ZGA through methods such as affecting Sebox and Lin28a in oocytes and embryos.

scholarly journals Maternal control of early embryogenesis in mammals

2015 ◽  
Vol 27 (6) ◽  
pp. 880 ◽  
Author(s):  
Kun Zhang ◽  
George W. Smith
Keyword(s):  
Maternal Effect ◽  
Oocyte Quality ◽  
Farm Animals ◽  
Maternal Control ◽  
Close Link ◽  
Embryonic Genome Activation ◽  
Ovarian Aging ◽  
Embryonic Genome ◽  
Maternal Effect Genes ◽  
Genome Activation

Oocyte quality is a critical factor limiting the efficiency of assisted reproductive technologies (ART) and pregnancy success in farm animals and humans. ART success is diminished with increased maternal age, suggesting a close link between poor oocyte quality and ovarian aging. However, the regulation of oocyte quality remains poorly understood. Oocyte quality is functionally linked to ART success because the maternal-to-embryonic transition (MET) is dependent on stored maternal factors, which are accumulated in oocytes during oocyte development and growth. The MET consists of critical developmental processes, including maternal RNA depletion and embryonic genome activation. In recent years, key maternal proteins encoded by maternal-effect genes have been determined, primarily using genetically modified mouse models. These proteins are implicated in various aspects of early embryonic development, including maternal mRNA degradation, epigenetic reprogramming, signal transduction, protein translation and initiation of embryonic genome activation. Species differences exist in the number of cell divisions encompassing the MET and maternal-effect genes controlling this developmental window. Perturbations of maternal control, some of which are associated with ovarian aging, result in decreased oocyte quality.

The Phenotype of mes-2, mes-3, mes-4 and mes-6, Maternal-Effect Genes Required for Survival of the Germline in Caenorhabditis elegans, Is Sensitive to Chromosome Dosage

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 167-185 ◽  
Author(s):  
Carol Garvin ◽  
Richard Holdeman ◽  
Susan Strome
Keyword(s):  
Gene Expression ◽  
Germ Cells ◽  
Maternal Effect ◽  
Early Embryo ◽  
Male Offspring ◽  
X Chromosomes ◽  
Control Of Gene Expression ◽  
Maternal Effect Genes ◽  
Surprising Finding

AbstractMutations in mes-2, mes-3, mes-4, and mes-6 result in maternal-effect sterility: hermaphrodite offspring of mes/mes mothers are sterile because of underproliferation and death of the germ cells, as well as an absence of gametes. Mutant germ cells do not undergo programmed cell death, but instead undergo a necrotic-type death, and their general poor health apparently prevents surviving germ cells from forming gametes. Male offspring of mes mothers display a significantly less severe germline phenotype than their hermaphrodite siblings, and males are often fertile. This differential response of hermaphrodite and male offspring to the absence of mes+ product is a result of their different X chromosome compositions; regardless of their sexual phenotype, XX worms display a more severe germline phenotype than XO worms, and XXX worms display the most severe phenotype. The sensitivity of the mutant phenotype to chromosome dosage, along with the similarity of two MES proteins to chromatin-associated regulators of gene expression in Drosophila, suggest that the essential role of the mes genes is in control of gene expression in the germline. An additional, nonessential role of the mes genes in the soma is suggested by the surprising finding that mutations in the mes genes, like mutations in dosage compensation genes, feminize animals whose male sexual identity is somewhat ambiguous. We hypothesize that the mes genes encode maternally supplied regulators of chromatin structure and gene expression in the germline and perhaps in somatic cells of the early embryo, and that at least some of their targets are on the X chromosomes.

scholarly journals Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection

2021 ◽  
Vol 102 (9) ◽  
Author(s):  
Constantina Borg ◽  
Aminu S. Jahun ◽  
Lucy Thorne ◽  
Frédéric Sorgeloos ◽  
Dalan Bailey ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Persistent Infection ◽  
Reverse Genetics ◽  
Viral Fitness ◽  
Murine Norovirus ◽  
Viral Loads ◽  
Immunocompetent Mice

Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and demonstrated its role as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Using reverse genetics, we generated MNV-3 viruses carrying a single or a triple termination codon inserted in the VF1 ORF. VF1-deleted MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. C57BL/6 mice infected with VF1-deleted MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a 3 month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonize the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilize multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

scholarly journals Causality analysis detects the regulatory role of maternal effect genes in the early Drosophila embryo

Genomics Data ◽  
2017 ◽  
Vol 11 ◽  
pp. 20-38 ◽  
Author(s):  
Zara Ghodsi ◽  
Xu Huang ◽  
Hossein Hassani
Keyword(s):  
Maternal Effect ◽  
Drosophila Embryo ◽  
Regulatory Role ◽  
Causality Analysis ◽  
Early Drosophila Embryo ◽  
Maternal Effect Genes

scholarly journals Double maternal effect: duplicated nucleoplasmin 2 genes,npm2aandnpm2b, are shared by fish and tetrapods, and have distinct and essential roles in early embryogenesis

2017 ◽  
Author(s):  
Caroline T. Cheung ◽  
Jérémy Pasquier ◽  
Aurélien Bouleau ◽  
Thao-Vi Nguyen ◽  
Franck Chesnel ◽  
...  
Keyword(s):  
Maternal Effect ◽  
Early Embryogenesis ◽  
Zygotic Genome Activation ◽  
Specific Expression ◽  
Wide Range ◽  
Maternal Effect Genes ◽  
Local Duplication ◽  
Functional Analyses ◽  
Genome Activation ◽  
Zygotic Genome

AbstractNucleoplasmin 2(npm2) is an essential maternal-effect gene that mediates early embryonic events through its function as a histone chaperone that remodels chromatin. Here we report the existence of twonpm2(npm2aandnpm2b) genes in zebrafish. We examined the evolution ofnpm2aandnpm2bin a variety of vertebrates, their potential phylogenetic relationships, and their biological functions using knockout models via the CRISPR/cas9 system. We demonstrated that the twonpm2duplicates exist in a wide range of vertebrates, including sharks, ray-finned fish, amphibians, and sauropsids, whilenpm2awas lost in Coelacanth and mammals, as well as some specific teleost lineages. Using phylogeny and synteny analyses, we traced their origins to the early stages of vertebrate evolution. Our findings suggested thatnpm2aandnpm2bresulted from an ancient local gene duplication, and their functions diverged although key protein domains were conserved. We then investigated their functions by examining their tissue distribution in a wide variety of species and found that they shared ovarian-specific expression, a key feature of maternal-effect genes. We also showed that bothnpm2aandnpm2bare maternally-inherited transcripts in vertebrates. Moreover, we used zebrafish knockouts to demonstrate thatnpm2aandnpm2bplay essential, but distinct, roles in early embryogenesis.npm2afunctions very early during embryogenesis, at or immediately after fertilization, whilenpm2bis involved in processes leading up to or during zygotic genome activation. These novel findings will broaden our knowledge on the evolutionary diversity of maternal-effect genes and underlying mechanisms that contribute to vertebrate reproductive success.Author SummaryThe protein and transcript of thenpm2gene have been previously demonstrated as maternal contributions to embryos of several vertebrates. Recently, twonpm2genes, denoted here asnpm2aandnpm2b, were discovered in zebrafish. This study was conducted to explore the evolutionary origin and changes that occurred that culminated in their current functions. We found that an ancient local duplication of the ancestralnpm2gene created the current two forms, and while most vertebrates retained both genes, notably, mammals and certain species of fish lostnpm2aand, albeit rarely, bothnpm2aandnpm2b. Our functional analyses showed thatnpm2aandnpm2bhave diverse but essential functions during embryogenesis, asnpm2amutants failed to undergo development at the earliest stage whilenpm2bmutants developed, although abnormally, until the zygotic genome activation stage after which their development was arrested followed subsequently by death. Our study is the first to clearly demonstrate the evolution, diversification, and functional analyses of thenpm2genes, which are essential maternal factors that are required for proper embryonic development and survival.

Digital x-ray mapping of nanometer-size supported bimetallic catalysts

1988 ◽  
Vol 46 ◽  
pp. 530-531
Author(s):  
L. T. Germinario
Keyword(s):  
Background Radiation ◽  
Metal Cluster ◽  
Single Element ◽  
Beam Diameter ◽  
X Rays ◽  
X Ray ◽  
Major Interest ◽  
Induced Changes

Understanding the role of metal cluster composition in determining catalytic selectivity and activity is of major interest in heterogeneous catalysis. The electron microscope is well established as a powerful tool for ultrastructural and compositional characterization of support and catalyst. Because the spatial resolution of x-ray microanalysis is defined by the smallest beam diameter into which the required number of electrons can be focused, the dedicated STEM with FEG is the instrument of choice. The main sources of errors in energy dispersive x-ray analysis (EDS) are: (1) beam-induced changes in specimen composition, (2) specimen drift, (3) instrumental factors which produce background radiation, and (4) basic statistical limitations which result in the detection of a finite number of x-ray photons. Digital beam techniques have been described for supported single-element metal clusters with spatial resolutions of about 10 nm. However, the detection of spurious characteristic x-rays away from catalyst particles produced images requiring several image processing steps.

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