Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus.

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.     

Kinetic model of DNA replication in eukaryotic organisms

We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition. The model describes well a large amount of different data within a simple theoretical framework. This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis. In particular, we have determined the frequency of origin activation in time and space during the cell cycle. Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast.     

Novel double promoter approach for identification of transgenic animals: A tool for in vivo analysis of gene function and development of gene-based therapies

Advances in vertebrate genetics have allowed studies of gene function in developing animals through gene knockout and transgenic analyses. These advances have encouraged the development of gene-based therapies through introduction of exogenous genes to enhance and/or replace dysfunctional or missing genes. However, in vertebrates, such analyses often involve tedious screening for transgenic animals, such as PCR-based genotype determinations. Here, we report the use of double-promoter plasmids carrying the transgene of interest and the crystallin-promotor-driven Green fluorescent protein (GFP) in transgenic Xenopus laevis tadpoles. This strategy allows a simple examination for the presence of GFP in the eyes to identify transgenic animals. PCR-based genotyping and functional characterization confirms that all animals expressing GFP in the eyes indeed carry the desired promoter/transgene units. Thus, the use of this and other similar vectors should dramatically improve current transgenesis protocols and reduce the time and cost for identifying transgenic animals.     

Retina dorsal/ventral patterning by Xenopus TBX3.

Although it is well known that patterning in the retina of vertebrates is essential for retina formation and for the retinotopic projection of axons in the embryo, knowledge of molecular and cellular mechanisms of retina patterning is limited. We have previously identified the Xenopus Tbx3 gene (XTbx3) which is expressed in the dorsal retina but not in the ventral retina in Xenopus embryos [H. Li, C. Tierney, L. Wen, J. Y. Wu, and Y. Rao (1997) Development 124, 603-615; M.-L. He, L. Wen, C. E. Campbell, J. Y. Wu, and Y. Rao (1999) Proc. Natl. Acad. Sci. USA 96, 10212-10217]. Dosage-sensitive phenotypes in humans suggest that the manipulation of the amount and location of its products could be informative for understanding its normal function. Here we report that ectopic expression of Tbx3 by mRNA injection suppressed formation of the ventral retina. Furthermore, Tbx3 injection led to inhibition of molecular markers for the ventral retina including Pax-2 and netrin, indicating that Tbx3 plays an important role in retina dorsal/ventral patterning in vertebrates by inhibition of gene expression for ventral retina specification.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

How we are shaped: the biomechanics of gastrulation

Although it is rarely considered so in modern developmental biology, morphogenesis is fundamentally a biomechanical process, and this is especially true of one of the first major morphogenic transformations in development, gastrulation. Cells bring about changes in embryonic form by generating patterned forces and by differentiating the tissue mechanical properties that harness these forces in specific ways. Therefore, biomechanics lies at the core of connecting the genetic and molecular basis of cell activities to the macroscopic tissue deformations that shape the embryo. Here we discuss what is known of the biomechanics of gastrulation, primarily in amphibians but also comparing similar morphogenic processes in teleost fish and amniotes, and selected events in several species invertebrates. Our goal is to review what is known and identify problems for further research.     

The golden anniversary of cloning: a celebratory essay

May 2002 marked the golden anniversary of the first cloned tadpoles. We celebrate this anniversary, as nuclear transplantation of frog cells into enucleated eggs became the prototype for cloning insects, fish, and mammals. We briefly review the salient results from amphibian cloning. Extension of these studies to mammalian species led to cloning adult cells, important advances in understanding nuclear reprogramming, and the construction of transgenic clones for biomedical applications. In addition, murine cloning clarified two problems unresolved in frog cloning: the unequivocal demonstration that nuclei of fully differentiated cells can direct the formation of fertile adults, and that abnormal expression of genes was responsible for the endoderm and neural syndromes in Rana clones.