Thylacine 1 is expressed segmentally within the paraxial mesoderm of the xenopus embryo and interacts with the notch pathway. We have analysed the expression of genes normally expressed in ectoderm, mesoderm or endoderm in individual cells from early and late gastrula embryos, by both in situ hybridization and singlecell rtpcr. Early xenopus laevis development involves highly complex morphogenic movements. Embryonic exposure to propylthiouracil disrupts leftright.
One of the inducible mrnas encodes a homeodomaincontaining protein that. The reader is thus provided with a histological presentation of immaculate quality. After an informative introduction to the early development of xenopus laevis follows the highlight of the book, namely the 42 plates, each consisting of at least four high resolution micrographs. Morphology of xenopus embryos and adults chapter 3. The activation of embryonic transcription of the xigf1 receptor during the early neurula stages further suggests that the xigf1 receptor may be involved. The ones marked may be different from the article in the profile. Expression of hace1 during xenopus laevis development was analyzed by wholemount in situ hybridization using an antisense probe upper panels and sense probe as a control lower panels.
Xenopus tropicalis as a model organism for genetics and genomics. The accumulation of ago proteins increases after mbt, but the identities. The cement gland is an ectodermal organ in the head of frog embryos, lying anterior to any neural tissue. Monsoroburq ah 2007 a rapid protocol for wish on xenopus embryos. Histological analysis had identified a phase of rapid assembly and remodeling of a fibronectin. Aug 11, 2017 early xenopus laevis embryogenesis is a powerful system to study mechanisms of developmental timing. Detection of the xvent2 transcription factor in early.
Jul 15, 2006 development of the primary mouth in xenopus laevis this region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. Benjamin peng and early development of xenopus laevis, a laboratory manual 2000, edited by. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the. The small, lidless eyes are located dorsally and turned upward stebbins, 2003. Patterned femtosecondlaser ablation of xenopus laevis. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and nonautonomous properties. Spatial expression of xenopus laevis hace1 in early development. S4, in agreement with observations made in xenopus tropicalis armisen et al. Developmentally regulated activation of apoptosis early in xenopus gastrulation results in cyclin a degradation during interphase of the cell cycle. Eye development and maturation in xenopus is a slow process approximately 60 hours in xenopus, calculated from data in and may allow us to more precisely identify how and in what cell types a circadian system oscillator controlling clock controlled genes is assembled in a specific tissue during development. This chapter describes in detail the construction and maintenance of a xenopus laevis colony. Sive hl, grainger rm, harland rm 2000 early development of xenopus laevis.
The manual features primers on live imaging of a variety of standard model organisms including c. The word xenopus means strange foot and laevis means smooth. The overall process of cell sorting was found to morphologically consist of two steps, concentrification and polarization, as designated here. The xenopus oocyte is a specialized single cell of colossal size 1 mm diameter that is highly amenable for microinjection and a stalwart model for heterologous expression. The cell sorting process of xenopus gastrula cells progresses. African clawed frog xenopus laevis species profile. Cold spring harbor laboratory press 2000, chapter 2, figure 2. As analyzed by specific rna expression, cement gland, like neural tissue, was induced by the dorsal mesoderm. Embryos were processed using a slight modification of the xenopus immunohistochemistry protocol from early development of xenopus laevis. At the beginning of lab, the instructor gives a short 10 minute powerpoint presentation introducing the benefits of xenopus laevis as a model organism as well as the threedimensional structure of the early cleavage and gastrula stage embryos using images and timelapse videos supporting file s8.
Microinjected xwnt8 mrna was able to rescue the development of a dorsally complete anteriorposterior axis in embryos ventralized by exposure to uv light. Transgenic xenopus laevis strain expressing cre recombinase. It covers xenopus husbandry, facility design and construction, necessary maintenance protocols, and monitoringdisaster recovery procedures. Stem celllike xenopus embryonic explants to study early.
It is written by several of the fields most prominent investigators in the light of the experience they gained as instructors in an intensive laboratory course taught at cold spring harbor laboratory since 1991. Manuscripts for books and for journal publication are invited from scientists world wide. Xenopus laevis is an attractive model to analyze gene function in development and organogenesis of vertebrates, as embryonic development occurs outside the female and the developing larvae are highly transparent, allowing an easy observation and manipulation of the organism during development sive et al. Activation of mapk in young embryos is abolished by injection of a dominant negative fgf receptor xfd rna, suggesting that mapk is. Early development of xenopus laevis a laboratory manual created date. Apoptosis is required during early stages of tail regeneration in xenopus laevis. The advent of xenopusbased pregnancy tests for human chorionic gonadotropin hcg in the 1930s and 1940s milton, 1946 led to the adoption of xenopus as a laboratory model species and was a boon. Single blastomere expression profiling of xenopus laevis embryos. Ectopic expression of the protooncogene int1 in xenopus. Sparrow db, jen wc, kotecha s, towers n, kintner c, mohun tj.
Digitized images and developmental data from nieuwkoop and faber 1994 normal table of xenopus laevis daudin. Development of the primary mouth in xenopus laevis deepdyve. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between xenopus and humans, allowing for the study of human diseasecausing genes. Pax3 and zic1 drive induction and differentiation of. A laboratory manual, a new volume in cold spring harbor laboratory press imaging series, presents a comprehensive set of essential visualization methods. The e3 ubiquitin ligase hace1 is required for early embryonic. Embryos develop into apparently normal gastrulae, but almost all surviving neurulae have a. While there is convincing evidence implicating drosophila int1 in pattern regulation, the normal role of int1 in vertebrate development is unclear. These examine important topics in molecular biology, genetics, development, virology, neurobiology, immunology and cancer biology. And of all the amphibians available for study, the most valuable is xenopus laevis,which is easy to keep and ovulates at any time of year in response to simple hormone injections. Xenopus laevis was chosen as an experimental model because of the wide knowledge of its developmental biology and because amphibian has found to represent key group in cb1 evolution through vertebrates 20, 21. The african clawed frog xenopus laevis, also known as the xenopus, african clawed toad, african clawtoed frog or the platanna is a species of african aquatic frog of the family pipidae. Establishing and maintaining a xenopus laevis colony for. Characterization of xznf1 in the early development of.
Differential gene expression during xenopus laevis development. Using xenopus as a model system for an undergraduate laboratory course in vertebrate development at the university of bordeaux, france. A laboratory manual cold spring harbor press, cold spring harbor, ny. Xenopus laevis is a dorsoventrally flattened frog with a relatively small head and a svl snoutvent length of 50over 140 mm 2over 5 in passmore and carruthers, 1995. The transcription factor xvent2 is thought to appear in xenopus embryos after activation of the zygote genome in late blastula. Amphibian embryos are supremely valuable in studies of early vertebrate development because they are large, handle easily, and can be obtained at many interesting stages. Its name is derived from the three short claws on each hind foot, which it uses to tear apart its food. The wild caught animals in our laboratory were the first to be used in a. Both nuclear size and dna amount contribute to midblastula. We suggest using a current version of chrome, firefox, or safari. Patterned femtosecondlaser ablation of xenopus laevis melanocytes for studies of cell migration, wound repair, and developmental processes jessica p. Eye development and maturation in xenopus is a slow process approximately 60 hours in xenopus, calculated from data in and may allow us to more precisely identify how and in what cell types a circadian system oscillator controlling clock controlled genes is assembled in a.
Pdf using xenopus as a model system for an undergraduate. Sherril green, dmv, phd, author the laboratory xenopus sp. Sive h, grainger r and harland r 2000 early development of xenopus laevis. Single blastomere expression profiling of xenopus laevis. Two key movements are gastrulation, which establishes germ layer spatial arrangement, and neurulation, which results in the folding and closure of the neural tube. The e3 ubiquitin ligase hace1 is required for early. Early xenopus laevis embryogenesis is a powerful system to study mechanisms of developmental timing. Limiting ago protein restricts rnai and microrna biogenesis. The material that xenopus can offer in biology is great in terms of quality and quantity. It describes the structure of the embryo and the locations, movements, and changes in shape and position of the major prospective tissues through the blastula, gastrula, and neurula stages. Everyday low prices and free delivery on eligible orders. In our preliminary experiment to search for target genes of secreted developmental regulators chordin and wnt8 in xenopus laevis unpublished data, we found that cnrip1 is a potential candidate for them.
Expression of activated map kinase in xenopus laevis. This cited by count includes citations to the following articles in scholar. To visualize fibril dynamics at these stages, we turned to using well. A laboratory manual 2nd revised edition by sive, hazel l. While the amphibian model xenopus laevis has had a very distinguished. Xenopus laevis embryos are an established model for studying kidney development. This chapter provides a practical guide to understanding and manipulating the early development of xenopus laevis, the african clawed frog. The cell sorting process of xenopus gastrula cells. Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Expression cloning from a pool of gastrula cdnas identified the wnt family member xwnt8 as having dorsal axisinducing activity in xenopus embryos. During blastula and gastrula stages of xenopus development, cells become progressively and asynchronously committed to a particular germ layer. Observations and experiments in the living frog embryo xenopus laevis excerpted and modified from.
Recent updates to the database include the xenopus laevis genome, a new xenopus tropicalis genome build, epigenomic data, collections of rna and protein sequences associated with genes, more powerful gene expression searches, a community and curated wiki, an extensive set of manually annotated gene expression patterns and a new database module. Nieuwkoop pd, faber j 1994 normal table of xenopus laevis garland, new york, 3rd ed. Genes free fulltext transgenic xenopus laevis line for. This collection of protocols for the study of early development in xenopus embryos is for investigators interested in the. Pdf heterologous protein expression in the xenopus oocyte. Here, we characterize change in transmembrane voltage potential vmem as a novel biophysical signal for eye induction in xenopus laevis. Oocytes are very rich in all the necessary rna and proteins for the first stages of development until early embryogenesis, i. In this manuscript protocols are described for the use of xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. This manual contains a comprehensive collection of protocols for the study of early. You can read early development of xenopus laevis a laboratory manual online using button below. Chromatin accessibility and histone acetylation in the. Cold spring harbor laboratory, cold spring harbor fgfr3 and regionalization of the anterior neural tube in zebrafish jan 2000. A blocking reaction was run using tbt with 2 mgml bsa and 20% heat.
An antibody specific for the activated form of mitogenactivated protein kinase mapk was used to monitor fgf signaling in vivo during early xenopus development. This conclusion is based on detection of the xvent2 mrna in embryos. Observations and experiments in the living frog embryo. Apr 02, 20 defining which key factors control commitment of an embryonic lineage among a myriad of candidates is a longstanding challenge in developmental biology and an essential prerequisite for developing stem cellbased therapies. After fertilization, the embryo undergoes twelve rapid. Targeted mutagenesis of multiple and paralogous genes in. Single blastomere expression profiling of xenopus laevis embryos of 8 to 32cells reveals developmental asymmetry. Recombinant xvent2 and a specific antiserum were obtained and used to study xvent2 content at various stages of early development of x. Function and regulation of foxf1 during xenopus gut development.
Chapter 5 early embryonic development of xenopus laevis. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres. For example, one frog ovary corresponds to mouse ovaries and western blots can be performed with protein samples from just one oocyte or 1 ul of extract see below. Richard m harland amphibian embryos are valuable in studies of early vertebrate development. The differential response to wnt signaling during early stages of xenopus development has been examined in greater depth recently. Even though xenopus is one of the two most popular nonmammalian animals used in biomedical research, their value in the lab suffers from a lack of standardization regarding their optimal care, breeding, and housing. Effect of water hardness on oocyte quality and embryo development in the african clawed frog xenopus laevis earl w. Molecular cloning and characterization of xenopus insulin.
Commitment implies that the induced cells not only express early lineage markers but further undergo an autonomous differentiation into the lineage. Interestingly, mesoderm with the highest cement glandinducing potential lay posterior to the ectoderm fated to form this organ, indicating that its induction occurred at a. Animal pole cells ac and vegetal pole cells vc dissociated from early xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. Injected rna is translated and the protein widely distributed. Jun 17, 2005 embryos were processed using a slight modification of the xenopus immunohistochemistry protocol from early development of xenopus laevis. Xenopus embryos are also amenable to largescale screening, but studies of kidney diseaserelated genes have been impeded because assessment of. Facility components and management protocols described within this chapter are based on our experience with the.
Omenetto1,3, 1department of biomedical engineering, tufts university, 4 colby street, medford, ma 02155, usa 2department of biology and tufts center for. Early development of xenopus laevis a laboratory manual. Studying in vivo retinal progenitor cell proliferation in. First imported to europe from south africa as a novelty in the late 19th century, these frogs survived and bred readily in. Xenopus laevis hace1 is mainly expressed in the ectoderm during early embryogenesis. Multiple signaling pathways are involved in regulating cell adhesion, migration, shape and polarity. Godfrey, phd1, and george e sanders, dvm2,3 husbandry and health of the african clawed frog, xenopus laevis, greatly influences the quality of oocytes produced. Fgf signaling has been implicated in germ layer formation and axial determination. We notice that you are using internet explorer, which is not supported by xenbase and may cause the site to display incorrectly.
Finally, the biphasic expression of xigf1 receptor suggests that, like in mammals 29, 30, the xigf1 receptor may play an important role in early embryonic development in xenopus laevis. Circadian genes are expressed during early development in. Endocannabinoid system in xenopus laevis development. In frogs, mesoderm presumably derives from presumptive ectoderm by induction under the control of diffusible substances produced by the endoderm. Perturbation of organogenesis by the herbicide atrazine in. Injected xwnt8 rna acts early in xenopus embryos to. Sive, hazel author, grainger, robert m author, harland, richard m author. After fertilization, the embryo undergoes twelve rapid synchronous cell divisions to reach the. To analyze the early phase of mesoderm induction, i have isolated cdna copies of mrnas induced in presumptive ectoderm by mesoderm inducing factor secreted by the xtc cell line. A genomewide approach identified 123 genes that are enriched on the dorsal side of gastrula stage embryos and are activated by the maternal wnt. To investigate a role of hace1 in early embryonic development, we first cloned a xenopus laevis hace1 homolog accession number ab894419 with a 2739 bp open reading frame. We have injected xenopus eggs with mouse int1 rna and monitored subsequent development.
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