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Cold Spring Harbor Protocols

Anthony L Mescher, Anton W Neff
A basic protocol is given for animal maintenance and surgery in studies of hindlimb regeneration in larval Xenopus laevis Unlike urodele limbs, those of larval frogs typically show much more variation in the extent of regeneration after amputation. Such variation can be reduced by optimizing the conditions of larval maintenance to regulate the rates of growth and development, by selecting only larvae with normal rates of growth and morphological development for experimental use, and by attention to precision and consistency in the proximo-distal level of surgical amputation...
June 12, 2018: Cold Spring Harbor Protocols
Nicolas Buisine, Xiaoan Ruan, Yijun Ruan, Laurent M Sachs
Proper gene expression involves communication between the regulatory elements and promoters of genes. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity through DNA looping between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing is a powerful high-throughput method for detection of genome-wide chromatin interactions...
June 12, 2018: Cold Spring Harbor Protocols
Jingjing Li, Enrique Amaya
The African clawed frog Xenopus has remarkable capacities to heal wounds rapidly and to regenerate complex tissues. Because of its experimental tractability, studies using Xenopus oocytes, embryos, and larvae have contributed extensively to our understanding of the molecular and cellular mechanisms underpinning wound healing and tissue regeneration. In this protocol, we describe wound-healing assays following mechanical or laser injuries of oocytes and multicellular epithelia in Xenopus laevis embryos. We also explain how to perform assays aimed at investigating the cellular and molecular events during wound healing, including gene knockdown and overexpression experiments...
June 12, 2018: Cold Spring Harbor Protocols
Nicolas Buisine, Xiaoan Ruan, Yijun Ruan, Laurent M Sachs
Proper gene expression involves communication between the regulatory elements and promoters of genes. Because regulatory elements can be located over a large range of genomic distances (from as close as a few hundred bp to as much as several Mb away), contact and communication between regulators and the core transcriptional machinery at promoters are mediated through DNA looping. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity between enhancer(s) and their target promoter(s)...
June 12, 2018: Cold Spring Harbor Protocols
Jonathan J Henry, Kimberly J Perry, Paul W Hamilton
Some vertebrates are able to regenerate the lens following its removal. This includes species in the genus Xenopus (i.e., X. laevis , X. tropicalis , and X. borealis ), the only anurans known to undergo lens regeneration. In Xenopus the regenerated lens is derived de novo from cells located within the basal-most layer of the larval corneal epithelium, and is triggered by factors provided by the neural retina. In larval frogs the corneal epithelium is underlain by an endothelium separated from the corneal epithelium except for a small central attachment (i...
June 12, 2018: Cold Spring Harbor Protocols
Jonathan J Henry, Kimberly J Perry, Paul W Hamilton
Lens regeneration can be studied in whole animals following removal of the original lens (lentectomy). However, culturing a whole animal can be impractical for assays involving small molecule inhibitors or proteins. Ex vivo eye tissue culture is an alternative approach for examining lens regeneration. The ex vivo culture system offers certain advantages when compared to the in vivo regeneration assay, as the percentage of cases showing lens differentiation can exceed that seen in whole animals. This culture system also allows for the treatment of eye tissues in small volumes, which helps ensure reproducibility and reduces the amount (and cost) of small-molecule inhibitors or exogenous proteins, etc...
June 12, 2018: Cold Spring Harbor Protocols
Reyna I Martinez-De Luna, Michael E Zuber
Genetically controlled cell type-specific ablation provides a reproducible method to induce regeneration that can be temporally and spatially controlled. Until recently, regeneration studies in Xenopus have relied on surgical methods to stimulate regeneration. These methods are labor intensive and not as reproducible as a genetically controlled approach. In this protocol we describe selective ablation of rod photoreceptors in the premetamorphic Xenopus laevis retina using the nitroreductase/metronidazole (NTR/Mtz) system...
May 22, 2018: Cold Spring Harbor Protocols
Sally A Moody
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...
May 16, 2018: Cold Spring Harbor Protocols
Gary S McDowell, Anna Philpott
The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts.
May 16, 2018: Cold Spring Harbor Protocols
Kevin S Dingwell, James C Smith
The animal cap explant is a simple but adaptable tool available to developmental biologists. The use of animal cap explants in demonstrating the presence of mesoderm-inducting activity in the Xenopus embryo vegetal pole is one of many elegant examples of their worth. Animal caps respond to a range of growth factors (e.g., Wnts, FGF, TGF-β), making them especially useful for studying signal transduction pathways and gene regulatory networks. Explants are also suitable for examining cell behavior and have provided key insights into the molecular mechanisms controlling vertebrate morphogenesis...
May 16, 2018: Cold Spring Harbor Protocols
Sally A Moody
Fate maps identify the precursors of an organ, and tracing the members of a blastomere lineage over time shows how its descendants come to populate that organ. The fates of the individual blastomeres of the two- to 32-cell Xenopus embryo have been fully mapped to reveal which cells are the major contributors to various cell types, tissues, and organs. However, because these fate maps were produced in the normal embryo, they do not reveal whether a precursor blastomere is competent to give rise to additional tissues or is already committed to its fate-mapped repertoire of descendants...
May 16, 2018: Cold Spring Harbor Protocols
Sara Mashoof, Breanna Breaux, Michael F Criscitiello
In jawed vertebrates from sharks to mammals, the thymus is the primary (or central) lymphoid tissue where T cells develop and mature. The particular stromal cell types, cytokine environment, and tissue organization in the thymus are essential for V(D)J recombination, positive selection for major histocompatibility complex recognition, and negative selection against self-peptide recognition of most αβ T cells. The thymectomy operation on Xenopus tadpole larva described here creates a T-cell-deficient model suitable for many immunology studies...
May 16, 2018: Cold Spring Harbor Protocols
Cristina Sáenz de Miera, Ethan Parr, Robert J Denver
In vivo gene transfer is a powerful tool for investigating protein function and gene regulation in living organisms. Delivery of plasmid DNA to the brain of Xenopus tadpoles by bulk electroporation-mediated (EM) gene transfer can be used to study the effects of ectopic gene expression on development, physiology, and behavior. It can also be used to mark cells for lineage tracing, investigate the in vivo function of gene regulatory elements when linked to a reporter gene, and introduce mutations into the genome of transfected cells, among other applications...
May 16, 2018: Cold Spring Harbor Protocols
Daniel R Buchholz, Yun-Bo Shi
Anuran metamorphosis resembles postembryonic development in mammals, a period around birth when many organs/tissues mature into their adult form as circulating thyroid and stress hormone levels are high. Unlike uterus-enclosed mammalian embryos, tadpoles develop externally and undergo the dramatic changes of hormone-dependent development totally independent of maternal influence, making them a valuable model in which to study vertebrate postembryonic organ development and maturation. Various protocols have been developed and/or adapted for studying metamorphosis in Xenopus laevis and X...
May 16, 2018: Cold Spring Harbor Protocols
Sally A Moody
The fates of individual cleavage-stage blastomeres and of groups of cells at the blastula through gastrula stages of Xenopus embryos have been mapped in great detail. These studies identified the major contributors of the three germ layers as well as a variety of tissues and organs and several specific cell types. One can use these fate maps to test the commitment of single cells or groups of cells to produce their normal repertoire of descendants, to identify the genes that regulate fate commitment, and to modulate the levels of gene expression in specific lineages to determine gene function in a variety of developmental processes...
May 16, 2018: Cold Spring Harbor Protocols
Kurt M Gibbs, Ben G Szaro
Axonal tracing allows visualizing connectivity between neurons, providing useful information about structure, neuronal location, and function of the nervous system. Identifying regenerating axons and their neuron cell bodies present the particular challenges of labeling the projections of interest while unambiguously demonstrating regrowth of those axons that have been damaged. In the developing brain, an additional labeling challenge arises, as new connections are being made throughout the duration of an experiment...
May 16, 2018: Cold Spring Harbor Protocols
Sally A Moody
The individual blastomeres of Xenopus two- to 32-cell embryos have been fate mapped. This work identified the precursors of most of the embryonic cell types, tissues and organs; however, the maps do not reveal the cell interactions or signaling pathways that are required for establishing cell fates. This protocol describes an explant culture approach for culturing blastomeres in isolation to test whether a cell's fate has been determined. Cleavage blastomeres can be cultured in a simple salt medium without added factors because they contain intracellular yolk platelets, which provide an intrinsic energy source...
May 16, 2018: Cold Spring Harbor Protocols
Riho Hasugata, Shinichi Hayashi, Aiko Kawasumi-Kita, Joe Sakamoto, Yasuhiro Kamei, Hitoshi Yokoyama
We describe a precise and reproducible gene-induction method in the amphibian, Xenopus laevis Tetrapod amphibians are excellent models for studying the mechanisms of three-dimensional organ regeneration because they have an exceptionally high regenerative ability. However, spatial and temporal manipulation of gene expression has been difficult in amphibians, hindering studies on the molecular mechanisms of organ regeneration. Recently, however, development of a Xenopus transgenic system with a heat-shock-inducible gene has enabled the manipulation of specific genes...
May 16, 2018: Cold Spring Harbor Protocols
Emilio E Méndez-Olivos, Juan Larraín
Mammals are not capable of regenerating their central nervous system (CNS); anamniotes, however, can regenerate in response to injury. The mechanisms that explain the different regenerative capabilities include: (i) extrinsic mechanisms that consider the cellular environment and extracellular matrix composition, (ii) intrinsic factors implicating the presence or absence of genetic programs that promote axon regeneration, and (iii) the presence or absence of neural stem and progenitors cells (NSPCs) that allow neurogenesis...
May 16, 2018: Cold Spring Harbor Protocols
Justin Sparks, Johannes C Walter
Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G1 phase of the cell cycle in that it supports replication licensing...
May 16, 2018: Cold Spring Harbor Protocols
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