Structural changes in oocyte nucleoli of Xenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II-III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II-III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II-III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4',6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II-III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble.     

Role of fibroblast growth factor during early midbrain development in Xenopus

Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain-hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signalling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. We report that explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signalling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. We propose that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF.     

Progesterone-induced DNA polymerase activity in full-grown oocytes of Xenopus laevis (1)

DNA polymerase activity increases in full-grown oocytes of Xenopus laevis during in vitro progesterone-induced maturation. This increase is inhibited by cycloheximide. The presence of the oocyte's nucleus (germinal vesicle) seems essential for the induction of this increase: in previously enucleated oocytes, the level of DNA polymerase activity does not change during progesterone treatment. Furthermore, a new form of DNA polymerase is detectable by DEAE chromatography in in vitro matured oocytes.     

Regulation of Raf-1-dependent signaling during early Xenopus development

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.     

Development of the gut in Xenopus laevis

The lining of the gut, together with the pancreas, liver, gall bladder, and respiratory system, is formed from the endoderm. The gut also contains smooth muscle and connective tissue of mesodermal origin. The amphibian Xenopus laevis is potentially an excellent model organism for studying how the cells of the endoderm and mesoderm become programmed to produce these internal organs. However, the anatomical complexity of the coiled gut presents a problem in studying its development. In order to overcome this problem we here present a comprehensive guide to the anatomy and histology of the developing Xenopus gut. We use a simple dissection to display its anatomy and the expression of four endodermal markers (alkaline phosphatase, IFABP, XlHbox8, and endodermin). We present schematic diagrams that show how the gut is arranged in three dimensions and how this organisation changes during development. We also present drawings of histological sections of the gut which allow any region to be identified and so represent an atlas for working with sections. Finally, we describe the histology of the cells of the various organs of the gut. This histological identification may be necessary for the identification of parts following experiments in which the normal pattern is disturbed.     

Control of gene expression in Xenopus early development

Risk adjustment is intended to minimize selection of patients or enrollees in health plans. Current efforts generally are recognized as inadequate, but improvement is difficult. The greatest short-term gain will come from introducing diagnostic information, though outpatient diagnosis data are unreliable. Initial efforts may use inpatient data, but this creates incentives to hospitalize people. Even exploiting diagnosis information leaves substantial imperfections. Partial capitation, common in behavioral health, reduces incentives to select patients and stent on services, but current policy resists it, perhaps because policymakers misinterpret the lesson of the Prospective Payment System. Theoretically, not paying plans more for providing additional services is optimal only if consumers are well informed.     

Development of the interphotoreceptor matrix in Xenopus laevis

Xenopus laevis interphotoreceptor matrix (IPM) contains a relatively aqueous insoluble wheat germ agglutinin (WGA)-binding component containing unidentified sialoglycoconjugates (Wood et al [1984] J. Comp. Neurol. 228:299-307). The appearance of WGA-binding macromolecules in the IPM was assessed during late embryonic stages (32-45) and in retinal rudiment cultures, using lectin cytochemistry and Western blotting techniques. Metabolic labeling of the neural retina versus retinal pigment epithelium (RPE)-choroid of juvenile Xenopus with 35S-MET was also evaluated in vivo and in vitro. Lectin cytochemistry of eyes from developmental stages 32-42 demonstrated distinct WGA-ferritin-binding sites on the developing outer segment membranes and in the IPM compartment. At stages 44-46 extensive WGA-binding domains were present as an extracellular network with other randomly scattered domains near the retinal pigment epithelium. Retinal rudiments from stage 32-33 were isolated and allowed to differentiate in hanging drop culture (Hollyfield and Witkowsky [1974] J. Exp. Zool. 189:357-377) with or without an investing pigment epithelium. Cultures developing with RPE exhibited an elaborate IPM with an anastomosing meshwork of WGA-ferritin binding sites. In the absence of RPE only limited amounts of binding restricted to the immediate vicinity of the developing photoreceptor outer segment membranes was observed. When Western blots were probed with WGA-HRP, stage 32-45 retinas demonstrated a major WGA-binding band of 126 kD. Similar amounts of WGA-binding macromolecules were synthesized in preparations cultured in the presence or absence of the investing RPE. During development the major WGA-binding component is a 126-kD protein. Equivalent synthesis of this protein in the presence and absence of RPE suggests that the PE is not required for synthesis of this 126-kD component. These results suggest that the retina is the primary site of synthesis of the WGA-binding components of the Xenopus IPM, whereas the PE plays a principal role in their assembly and organization.     

The effect of concanavalin-A on the reaggregation of cells dissociated from Xenopus laevis early embryos

The effects of concanavalin-A on the reaggregation and sorting of cells from Xenopus laevis early embryos have been studied. The results suggest that at high concentrations, concanavalin-A can prevent reaggregation.     

Larval development of tectal efferents and afferents in Xenopus laevis (Amphibia Anura)

The development of tectal connections in Xenopus laevis had been investigated using the degeneration technique to demonstrate the efferent pathways and the retrograde HRP transport to label the afferent pathways. Bilateral tectal efferents were present as soon as the beginning of metamorphosis. Ascending efferents originated from the anterior tectal part terminate in the secondary visual thalamic centres whereas the descending efferents coming from the posterior tectal part reached the tegmentum and the medulla oblongata. At this same time, the optic tectum already received secondary visual afferents originating in the ipsilateral pretectum and non-visual afferents from the ipsilateral semicircular torus and tegmentum. Some sparse bilateral isthmotectal connections were also present. Later, efferent pathways showed an increasing number of fibres whereas the sites of origin of afferents became more diversified: the dorsal thalamus, the suprachiasmatic area, the tegmental nuclei and in the medulla oblongata, the reticular and octavolateral areas sent bilateral projections to the optic tectum. At the end of metamorphosis, we noted ipsilateral olivotectal fibres and reciprocal connections between the tectum and the area of the Vth nerve. These last findings and the presence of the following direct projections, not previously reported in Anurans: the reciprocal connections between the tectum and the semicircular torus or the octavolateral area, underline the implication of the optic tectum in the multisensory (visual, acoustic, vibratory) integration elicited during the larval behavior. Also, the relations between the optic tectum and the lateral line system are particularly examined in the discussion.