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Author: Sophie Wiszniak Publisher: ISBN: Category : Germ cells Languages : en Pages : 192
Book Description
"In most organisms, the primordial germ cells are specified and set aside from the surrounding somatic tissues very early in development. Their ability to carry out a gene regulatory program quite distinct from the surrounding somatic cells, and their capacity to specify entire new organisms has made them a focus of many studies that seek to understand how specific transcriptional and translational programs contribute to cell fate. Zebrafish, a vertebrate with external development of the embryo, is currently one of the best animal models for understanding the molecular basis of germ cell specification. Briefly, germ cell specification is dependent on maternally provided cytoplasmic determinants, termed the germ plasm. The germ plasm, is localised to areas of the embryo that will become the germ cells later in development by inheritance the germ plasm through cleavage divisions. A number of mRNA components of the germ plasm have been identified; interestingly many of them encode RNA-binding proteins, and almost all of them have invertebrate and mammalian orthologues. Evidence suggests that these maternally provided mRNA determinants are specifically maintained in the germ cells throughout embryonic development, and at least some of these gene products are essential for germ cell specification. A number of studies have begun to elucidate the molecular mechanisms that allow germ cell specific maintenance of these mRNAs, and also to identify how maternally provided messages destined for the germ cells are destabilised and eliminated in the somatic tissues. For example, the germ cell specific mRNAs nanos and TDRD7 are destabilised in somatic cells through interactions of the 3 ́UTR sequences with the microRNA miR-430. This miR-430-mediated repression is overcome in germ cells through the binding of an RNA-binding protein Dead end (DND) to distinct sites within the nanos and TDRD7 3 ́UTRs. This thesis details a study of the zebrafish orthologue of HuB, a highly conserved RNAbinding protein with expression in neurons, testes and ovaries in adult vertebrates. In zebrafish, HuB mRNA is maternally provided, and is restricted to the germ cells by 24 hours of development; this is the first report to indicate expression of HuB in the germ cells of vertebrates, suggesting a possible role for HuB in germ cell development. Through detailed mutagenesis studies, the HuB 3 ́UTR has been found to contain a set of four destabilising elements, which bring about somatic degradation of the mRNA, and a separate, 30-nucleotide motif that is responsible for germ cell specific stabilisation of the message. None of these identified destabilising elements are targets for miR-430, and thus they represent novel sequence elements for somatic message degradation in zebrafish. Through a candidate screening approach, DAZL, a germ cell specific RNA-binding protein, was identified as being capable of stabilising HuB mRNA. Further-more, DAZL was shown to mediate this stabilisation of HuB mRNA by interacting, either directly or indirectly, with the 30-nucleotide stabilisation element that was indentified in the HuB 3 ́UTR. This elucidation of the mechanisms of germ cell specific expression of the HuB mRNA is an important finding, for it reveals mechanisms of post-transcriptional regulation that are distinct from that of other germ cell specific mRNAs. In summary, the identification of HuB as a germ cell specific mRNA, and the determination of the post-transcriptional mechanisms responsible for this specific expression is an important first step in understanding how HuB and other germ cell specific RNA-binding proteins contribute to germ cell development and function." -- leaf 3.
Author: Sophie Wiszniak Publisher: ISBN: Category : Germ cells Languages : en Pages : 192
Book Description
"In most organisms, the primordial germ cells are specified and set aside from the surrounding somatic tissues very early in development. Their ability to carry out a gene regulatory program quite distinct from the surrounding somatic cells, and their capacity to specify entire new organisms has made them a focus of many studies that seek to understand how specific transcriptional and translational programs contribute to cell fate. Zebrafish, a vertebrate with external development of the embryo, is currently one of the best animal models for understanding the molecular basis of germ cell specification. Briefly, germ cell specification is dependent on maternally provided cytoplasmic determinants, termed the germ plasm. The germ plasm, is localised to areas of the embryo that will become the germ cells later in development by inheritance the germ plasm through cleavage divisions. A number of mRNA components of the germ plasm have been identified; interestingly many of them encode RNA-binding proteins, and almost all of them have invertebrate and mammalian orthologues. Evidence suggests that these maternally provided mRNA determinants are specifically maintained in the germ cells throughout embryonic development, and at least some of these gene products are essential for germ cell specification. A number of studies have begun to elucidate the molecular mechanisms that allow germ cell specific maintenance of these mRNAs, and also to identify how maternally provided messages destined for the germ cells are destabilised and eliminated in the somatic tissues. For example, the germ cell specific mRNAs nanos and TDRD7 are destabilised in somatic cells through interactions of the 3 ́UTR sequences with the microRNA miR-430. This miR-430-mediated repression is overcome in germ cells through the binding of an RNA-binding protein Dead end (DND) to distinct sites within the nanos and TDRD7 3 ́UTRs. This thesis details a study of the zebrafish orthologue of HuB, a highly conserved RNAbinding protein with expression in neurons, testes and ovaries in adult vertebrates. In zebrafish, HuB mRNA is maternally provided, and is restricted to the germ cells by 24 hours of development; this is the first report to indicate expression of HuB in the germ cells of vertebrates, suggesting a possible role for HuB in germ cell development. Through detailed mutagenesis studies, the HuB 3 ́UTR has been found to contain a set of four destabilising elements, which bring about somatic degradation of the mRNA, and a separate, 30-nucleotide motif that is responsible for germ cell specific stabilisation of the message. None of these identified destabilising elements are targets for miR-430, and thus they represent novel sequence elements for somatic message degradation in zebrafish. Through a candidate screening approach, DAZL, a germ cell specific RNA-binding protein, was identified as being capable of stabilising HuB mRNA. Further-more, DAZL was shown to mediate this stabilisation of HuB mRNA by interacting, either directly or indirectly, with the 30-nucleotide stabilisation element that was indentified in the HuB 3 ́UTR. This elucidation of the mechanisms of germ cell specific expression of the HuB mRNA is an important finding, for it reveals mechanisms of post-transcriptional regulation that are distinct from that of other germ cell specific mRNAs. In summary, the identification of HuB as a germ cell specific mRNA, and the determination of the post-transcriptional mechanisms responsible for this specific expression is an important first step in understanding how HuB and other germ cell specific RNA-binding proteins contribute to germ cell development and function." -- leaf 3.
Author: Soma Dash Publisher: ISBN: 9780438423435 Category : Languages : en Pages : 156
Book Description
Eye development in vertebrates is initiated in late gastrulation and involves coordinated morphogenesis between the optic vesicle and the non-neural surface ectoderm resulting in the formation of the neural retina and the lens, respectively. While transcription and signaling events required for eye development are well understood, post-transcriptional control of gene expression, especially mediated by RNA-binding proteins (RBPs) is less clear. This represents a significant knowledge-gap as RBPs are important regulatory molecules in the cell that can control the fate of their target mRNAs by interacting with them throughout the mRNA life-cycle and mediating their processing, intra-cellular transport and localization, stability, translation into protein, and ultimately, their degradation. This is also a significant knowledge gap because there are similar number of RBPs encoded by the human genome as there are transcription factors, but the former class of proteins are not as well understood in the context of organogenesis and birth defects as compared to the latter. ☐ While high-throughput sequencing has identified several RBPs to be expressed in the eye, the functional significance in eye development for the vast majority of these factors is yet to be determined. Recently, the Lachke laboratory has identified two conserved RBPs required for eye development, Tdrd7 and Celf1, whose deficiency in the lens results in cataract in vertebrates. To further investigate the importance of RBP-mediated post-transcriptional gene expression control in eye development, I applied a systems-based bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to identify two new RBPs, Rbm24 and Caprin2, which are enriched during early mouse lens development, but whose molecular function in eye development had thus far not been determined. In this research dissertation, I have characterized the function of both Rbm24 and Caprin2 using constitutive and conditional targeted gene deletion mouse models. Further, in collaboration with Dr. Diane Slusarski’s laboratory (University of Iowa), zebrafish rbm24a knockout (by CRISPR/Cas9) and knockdown (by morpholino) mutants were generated and characterized. Together, these findings have led to a comprehensive understanding of the function of these RBPs in vertebrate eye development. ☐ Rbm24-targeted deletion in mouse and rbm24a-CRISPR/Cas9-targeted knockout or morpholino-knockdown in zebrafish causes the developmental defects microphthalmia (small eye) or anophthalmia (no eye). Rbm24 deficiency leads to apoptotic defects in the mouse ocular tissue as well as downregulation of eye development markers such as Sox2, Lhx2, Jag1, E-cadherin and g-Crystallins. Further, similar to the observations in the mouse, sox2 expression is also found to be reduced in rbm24a-morphant zebrafish, indicating the conservation of the Rbm24-Sox2 regulatory module in vertebrate eye development. About 20% of human anophthalmia cases are linked to SOX2 mutations alone. Therefore, I focused on investigating the post-transcriptional molecular mechanism of Rbm24-mediated Sox2 regulation. Sox2 is an intronless gene whose encoded mRNA contains AU-rich regions (ARE) in its 3’UTR. Interestingly, Rbm24 is known to bind to ARE sites in target mRNA. Therefore, to test if Rbm24 directly binds to Sox2 mRNA in vitro and in vivo, I performed RNA-Electrophoretic Mobility Shift assay (EMSA) and RNA-Immunoprecipitation (RIP), respectively. RNA-EMSA showed that Rbm24 protein directly binds to a 20 bp oligomer based on the mouse Sox2 mRNA sequence, and that an intact ARE is necessary for this protein-RNA binding. In turn, RIP assay on E14.5 wildtype mouse ocular tissue suggests that Rbm24 directly binds to Sox2 mRNA in vivo in eye development. To understand the biological significance of this direct Rbm24 protein-Sox2 mRNA molecular interaction, I performed an RNA-decay assay in NIH3T3 cells by co-transfected them with an Rbm24-overexpression vector and a Renilla luciferase reporter vector. In this assay, the Renilla luciferase gene ORF (open reading frame) is fused with the mouse Sox2 mRNA 3’UTR, which contains the three intact ARE sites, and reporter transcripts were quantified after Actinomycin-D treatment to transfected cells. This analysis demonstrates that in conditions of Rbm24 over-expression, the intact Sox2 3’UTR can render increased stability to the reporter transcript. Thus, Rbm24 positively controls Sox2 expression by binding to ARE sites in its 3’UTR and increasing its mRNA stability. Further, mutation analysis in the RNA-decay assay extends the in vitro observation that the binding of Rbm24 to the Sox2 mRNA 3’UTR depends on ARE by providing in vivo evidence that the presence of the ARE sites is necessary for the stability effect rendered by the Sox2 mRNA 3’UTR upon Rbm24 overexpression. Further, because Sox2 is one of the original four Yamanaka pluripotency/cellular reprogramming factor (along with Oct4, Klf4 and c-Myc), I investigated the impact of Rbm24 on the expression of other reprogramming factors such as Oct4, Klf4, c-Myc as well as, Nanog, another established pluripotency factor. I find that over-expression of Rbm24 in several different cell lines such as NIH3T3 (mouse embryo fibroblast cell line), 21EM15 (mouse lens epithelial cell line) and C2C12 (mouse myoblast cell line) results in the up-regulation of Sox2, Oct4 and Klf4. Further, in Rbm24-overexpressed C2C12 cells, Nanog and c-Myc are also upregulated. These data highlight that Rbm24 mediates post-transcriptional control of key transcription and pluripotency factors in vertebrate development. ☐ To gain insight into the function of the other newly identified RBP, Caprin2, in lens biology, I first performed expression analysis of Caprin2 in mouse lens development using in situ hybridization, western blotting and immunostaining. These experiments validate the iSyTE prediction that Caprin2 mRNA and protein are highly expressed and enriched in mouse embryonic and postnatal lens. I generated lens-specific Caprin2 conditional knockout (cKO) mouse mutants using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2cKO/cKO mice, wherein Caprin2 is expected to be deleted in the lens starting from E9.5 due to Cre-mediated re-arrangement of the Caprin2 alleles, revealed two distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2cKO/cKO mutants have an abnormally compact “lens nucleus”, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Further, at a reduced penetrance (8%), I find that Caprin2cKO/cKO mutants exhibit an ocular defect wherein the lens and the cornea remain attached by a persistent stalk, resembling the human developmental defect termed Peters anomaly. These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development. ☐ Together the findings in this dissertation have demonstrated that conserved RBPs such as Rbm24 and Caprin2 have evolved distinct functions in vertebrate eye development and their deficiency leads to microphthalmia and anophthalmia, and lens defects and Peters anomaly, respectively, thus impacting the study of ocular defects in humans.
Author: Rita Payan-Carreira Publisher: BoD – Books on Demand ISBN: 9535122681 Category : Medical Languages : en Pages : 254
Book Description
The chapters in this volume of "Insights from Animal Reproduction" address several, particular hot topics in the field of reproduction. The book begins with a comprehensive overview of the cryopreservation of sheep-produced embryos. The following chapter revises the assisted reproductive techniques available for South American wild mammals. Chapter 3 presents the technical procedures necessary to produce transgenic goats. Chapter 4 provides a comprehensive revision of the major molecular determinants of litter size in prolific species. Chapter 5 examines the germ cell determinant transmission, segregation, and function using the zebrafish as a model for germ cell specification in the embryo. Chapter 6 summarizes the current understanding of the molecular and cellular mechanisms regulating the early stages of folliculogenesis. Chapter 7 examines the sperm motility regulatory proteins as a tool to enhance sperm quality in cryopreservation processes. Chapter 8 discusses contemporary knowledge on the effects of extremely low frequency magnetic fields (ELF-MF) on male reproductive function in rodents. Chapter 9 highlights the importance of the cytogenetic evaluation in searching for causes of infertility of phenotypically normal animals, as well as individuals with an abnormal sex development. The last chapter provides evidence that other uterine diseases may be hidden behind the clinical diagnosis of pyometra that in some case may have a poor outcome.
Author: Derek J. Chadwick Publisher: John Wiley & Sons ISBN: 0470319429 Category : Science Languages : en Pages : 260
Book Description
The formation of blood vessels is an essential aspect of embryogenesis in vertebrates. It is a central feature of numerous post-embryonic processes, including tissue and organ growth and regeneration. It is also part of the pathology of tumour formation and certain inflammatory conditions. In recent years, comprehension of the molecular genetics of blood vessel formation has progressed enormously and studies in vertebrate model systems, especially the mouse and the zebrafish, have identified a common set of molecules and processes that are conserved throughout vertebrate embryogenesis while, in addition, highlighting aspects that may differ between different animal groups. The discovery in the past decade of the crucial role of new blood vessel formation for the development of cancers has generated great interest in angiogenesis (the formation of new blood vessels from pre-existing ones), with its major implications for potential cancer-control strategies. In addition, there are numerous situations where therapeutic treatments either require or would be assisted by vasculogenesis (the de novo formation of blood vessels). In particular, post-stroke therapies could include treatments that stimulate neovascularization of the affected tissues. The development of such treatments, however, requires thoroughly understanding the developmental properties of endothelial cells and the basic biology of blood vessel formation. While there are many books on angiogenesis, this unique book focuses on exactly this basic biology and explores blood vessel formation in connection with tissue development in a range of animal models. It includes detailed discussions of relevant cell biology, genetics and embryogenesis of blood vessel formation and presents insights into the cross-talk between developing blood vessels and other tissues. With contributions from vascular biologists, cell biologists and developmental biologists, a comprehensive and highly interdisciplinary volume is the outcome.
Author: Florence Louise Marlow Publisher: Morgan & Claypool Publishers ISBN: 161504051X Category : Family & Relationships Languages : en Pages : 221
Book Description
Eggs of all animals contain mRNAs and proteins that are supplied to or deposited in the egg as it develops during oogenesis. These maternal gene products regulate all aspects of oocyte development, and an embryo fully relies on these maternal gene products for all aspects of its early development, including fertilization, transitions between meiotic and mitotic cell cycles, and activation of its own genome. Given the diverse processes required to produce a developmentally competent egg and embryo, it is not surprising that maternal gene products are not only essential for normal embryonic development but also for fertility. This review provides an overview of fundamental aspects of oocyte and early embryonic development and the interference and genetic approaches that have provided access to maternally regulated aspects of vertebrate development. Some of the pathways and molecules highlighted in this review, in particular, Bmps, Wnts, small GTPases, cytoskeletal components, and cell cycle regulators, are well known and are essential regulators of multiple aspects of animal development, including oogenesis, early embryogenesis, organogenesis, and reproductive fitness of the adult animal. Specific examples of developmental processes under maternal control and the essential proteins will be explored in each chapter, and where known conserved aspects or divergent roles for these maternal regulators of early vertebrate development will be discussed throughout this review. Table of Contents: Introduction / Oogenesis: From Germline Stem Cells to Germline Cysts / Oocyte Polarity and the Embryonic Axes: The Balbiani Body, an Ancient Oocyte Asymmetry / Preparing Developmentally Competent Eggs / Egg Activation / Blocking Polyspermy / Cleavage/ Mitosis: Going Multicellular / Maternal-Zygotic Transition / Reprogramming: Epigenetic Modifications and Zygotic Genome Activation / Dorsal-Ventral Axis Formation before Zygotic Genome Activation in Zebrafish and Frogs / Maternal TGF-β and the Dorsal-Ventral Embryonic Axis / Maternal Control After Zygotic Genome Activation / Compensation by Stable Maternal Proteins / Maternal Contributions to Germline Establishment or Maintenance / Perspective / Acknowledgments / References
Author: National Research Council Publisher: National Academies Press ISBN: 0309070864 Category : Nature Languages : en Pages : 348
Book Description
Scientific Frontiers in Developmental Toxicology and Risk Assessment reviews advances made during the last 10-15 years in fields such as developmental biology, molecular biology, and genetics. It describes a novel approach for how these advances might be used in combination with existing methodologies to further the understanding of mechanisms of developmental toxicity, to improve the assessment of chemicals for their ability to cause developmental toxicity, and to improve risk assessment for developmental defects. For example, based on the recent advances, even the smallest, simplest laboratory animals such as the fruit fly, roundworm, and zebrafish might be able to serve as developmental toxicological models for human biological systems. Use of such organisms might allow for rapid and inexpensive testing of large numbers of chemicals for their potential to cause developmental toxicity; presently, there are little or no developmental toxicity data available for the majority of natural and manufactured chemicals in use. This new approach to developmental toxicology and risk assessment will require simultaneous research on several fronts by experts from multiple scientific disciplines, including developmental toxicologists, developmental biologists, geneticists, epidemiologists, and biostatisticians.
Author: Roland Dosch Publisher: ISBN: 9781071609705 Category : Cell differentiation Languages : en Pages : 383
Book Description
This volume details a wide range of methods, ranging from beginner through advanced, used to further study zebrafish and fish germline. Chapter guide readers through cultivating and manipulating germ cells, imaging of germline processes and the molecular analysis of their, protein, and RNA. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Germline Development in the Zebrafish: Methods and Protocols aims to ensure successful results in the further study of this vital field.
Author: L. Joseph Su Publisher: Springer ISBN: 1447166787 Category : Medical Languages : en Pages : 327
Book Description
This book examines the toxicological and health implications of environmental epigenetics and provides knowledge through an interdisciplinary approach. Included in this volume are chapters outlining various environmental risk factors such as phthalates and dietary components, life states such as pregnancy and ageing, hormonal and metabolic considerations and specific disease risks such as cancer cardiovascular diseases and other non-communicable diseases. Environmental Epigenetics imparts integrative knowledge of the science of epigenetics and the issues raised in environmental epidemiology. This book is intended to serve both as a reference compendium on environmental epigenetics for scientists in academia, industry and laboratories and as a textbook for graduate level environmental health courses. Environmental Epigenetics imparts integrative knowledge of the science of epigenetics and the issues raised in environmental epidemiology. This book is intended to serve both as a reference compendium on environmental epigenetics for scientists in academia, industry and laboratories and as a textbook for graduate level environmental health courses.
Author: Lucy W. Barrett Publisher: Springer Science & Business Media ISBN: 3034806795 Category : Science Languages : en Pages : 63
Book Description
There is now compelling evidence that the complexity of higher organisms correlates with the relative amount of non-coding RNA rather than the number of protein-coding genes. Previously dismissed as “junk DNA”, it is the non-coding regions of the genome that are responsible for regulation, facilitating complex temporal and spatial gene expression through the combinatorial effect of numerous mechanisms and interactions working together to fine-tune gene expression. The major regions involved in regulation of a particular gene are the 5’ and 3’ untranslated regions and introns. In addition, pervasive transcription of complex genomes produces a variety of non-coding transcripts that interact with these regions and contribute to regulation. This book discusses recent insights into the regulatory roles of the untranslated gene regions and non-coding RNAs in the control of complex gene expression, as well as the implications of this in terms of organism complexity and evolution.
Author: Sophie Wiszniak
Author: Sophie Wiszniak
Author: Shannon Marie Byrd
Author: Soma Dash
Author: Rita Payan-Carreira
Author: Derek J. Chadwick
Author: Florence Louise Marlow
Author: National Research Council
Author: Roland Dosch
Author: L. Joseph Su
Author: Lucy W. Barrett