Expression of ras-like proteins in embryonic and adult cells of Xenopus laevis

A polyclonal antibody raised against v-Ha-ras p21 was purified and its specificity was checked on Ha-ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one-dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full-grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two-dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modifications of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two-dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.     

Kindlin-2 expression in adult tissues correlates with their embryonic origins

Kindlin-2 functions in the maintenance of homeostasis and in human diseases. This study investigated the interrelationship between Kindlin-2 expression in tissues and the corresponding germ layers from which these tissues originated. Kindlin-2 expression was examined in normal adult human organs and human cancer tissues by immunohistochemical analyses. Analysis of Kindlin-2 mRNA levels in adult human organs in the Oncomine dataset revealed Kindlin-2 is highly expressed in mesoderm-derived organs. However, Kindlin-2 was negative or weakly expressed in endoderm/ectoderm-derived organs. Interestingly, the abnormal expression of Kindlin-2 was observed in a variety of human cancers. In agreement with its expression profile in humans, Kindlin-2 was also highly expressed in mesoderm-derived organs in mouse embryos with the exception of strong Kindlin-2 expression in ectoderm-derived spinal cord and ganglia, tissues that are highly mobile during embryonic development. Importantly, we demonstrated the expression level of Kindlin-2 in adult organs correlated with their embryonic dermal origins and deregulation of Kindlin-2 in tissues is associated with tumor progression. This finding will help us understand the dual role of Kindlin-2 in the regulation of tumor progression and embryonic development.     

Experimental analysis of ventral blood island hematopoiesis in Xenopus embryonic chimeras

The frequencies and potentialities of hematopoietic stem cells from 20-hr-old Xenopus embryos were examined by transplanting cytogenetically distinct ventral blood island tissue from diploid to triploid embryos. Thirty-five-day-old larvae were examined for the presence of donor-derived cells in their erythrocyte, thymocyte, and B lymphocyte populations by analyzing DNA content using flow cytometry. These experiments demonstrated that B lymphocytes, as well as erythrocytes and thymocytes, were derived from the ventral blood island. Data obtained by transplanting graded sized pieces of ventral blood island suggested that restricted erythroid precursors were present within the region by 20 hr postfertilization. Differentiation of both B- and T-lymphoid precursors from small pieces of ventral blood island was markedly enhanced when this tissue was grafted onto peripheral areas within the blood island region. Analysis of these data using repopulation statistics suggested that circulating larval erythrocytes of ventral blood island origin were derived from six or seven precursors. Each lobe of the thymus was colonized by three precursors, one of which was ventral blood island derived.     

Anion transport in embryonic and adult chicken red blood cells

The rates of Cl and SO₄ transport at 0° and 37°C, respectively, have been measured under exchange conditions for red blood cells of embryonic and adult chickens. It was found that the rate of self-exchange of SO₄ in embryonic red cells decreases as the embryo matures, and that the SO₄ transport rate was lower in adult compared to embryonic red cells. In contrast, no difference in the rate of Cl self-exchange was found between adult and embryonic red cells.     

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

In vitro formation of neuromuscular junctions between adult Rana muscle fibres and embryonic Xenopus neurons

Adult muscle fibres of the frog Rana temporaria were cultured with neurons from embryos of the frog Xenopus laevis. Electron microscopical and electro-physiological examination of the cultures showed that hetero-specific (Xenopus-Rana) neuromuscular junctions were formed in vitro. Nerve processes, without any Schwann cell covering, made contacts anywhere along a muscle fibre, and the junctions resembled those seen during early regeneration of neuromuscular synapses in situ. Functional contacts, as inferred by the presence of spontaneous miniature endplate potentials, or currents, were more common if the muscle fibres were denervated prior to culturing with neurons. Miniature endplate currents (m.e.p.cs) had a skewed amplitude distribution, with many small events lost in the recording noise, and their mean amplitude was much smaller than that of m.e.p.cs in the original lumbricalis muscle. The time constant of decay of m.e.p.cs in the hetero-specific junctions formed in vitro was several times longer than the decay of m.e.p.cs in the original muscle. Analysis of membrane current noise elicited by ionophoretically applied acetylcholine (ACh) suggests that the slower decay of m.e.p.cs in the junctions formed in vitro is due to a prolonged lifetime of the channels opened by ACh and to repetitive activation of ACh-receptors, which becomes possible because of a comparative lack of cholinesterase in the junctions.     

Two transitions of haemoglobin expression in Xenopus: from embryonic to larval and from larval to adult

Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins (X. borealis) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.     

Distinct mechanisms control the timing of differentiation of two myeloid populations in Xenopus ventral blood islands

Previous study has suggested that distinct populations of myeloid cells exist in the anterior ventral blood islands (aVBI) and posterior ventral blood islands (pVBI) in Xenopus neurula embryo. However, details for differentiation programs of these two populations have not been elucidated. In the present study, we examined the role of Wnt, vascular endothelial growth factor (VEGF) and fibroblast growth factor signals in the regulation of myeloid cell differentiation in the dorsal marginal zone and ventral marginal zone explants that are the sources of myeloid cells in the aVBI and pVBI. We found that regulation of Wnt activity is essential for the differentiation of myeloid cells in the aVBI but is not required for the differentiation of myeloid cells in the pVBI. Endogenous activity of the VEGF signal is necessary for differentiation of myeloid cells in the pVBI but is not involved in the differentiation of myeloid cells in the aVBI. Overall results reveal that distinct mechanisms are involved in the myeloid, erythroid and endothelial cell differentiation in the aVBI and pVBI.     

Distinct molecular origins for Denys-Drash and Frasier syndromes

The direct involvment of the Wilm's tumor suppressor gene (WT1) in Denys-Drash syndrome through mutations within exons 8 or 9 has recently been established. The absence of such alterations in three patients with Frasier syndrome provides a molecular basis for distinguishing these two syndromes that are associated with streak gonads, pseudohermaphroditism and renal failure.     

Telomerase activity in germline and embryonic cells of Xenopus.

Telomerase is a ribonucleoprotein which synthesizes telomere repeats onto chromosome ends. Telomerase activity is involved in telomere length maintenance. We used Xenopus laevis as a model system to study the expression of telomerase activity in germline cells and during early development. We identified a non-processive telomerase activity in manually dissected nuclei of Xenopus stage VI oocytes. Telomerase activity was detected throughout oogenesis and embryogenesis. Telomerase was active in both S and M phase cell cycle extracts, suggesting that telomerase activity is not regulated with chromosomal DNA replication.     

Common origins of blood and blood vessels in adults?

After embryonic development, the vast majority of cells are differentiated and all organs are in place. Growth of the organism then ensues and continues until adulthood, whereupon cell division largely ceases. In some tissues, notably the bone marrow, skin, and gut, cell proliferation continues throughout life to replace cells lost by attrition. This regeneration is fueled by rare, long-lived, and largely quiescent stem cells that give rise to committed progenitors, which in turn generate large numbers of fully differentiated cells. Mounting evidence suggests that such cells can significantly contribute to tissue repair and regeneration in adults and may therefore prove beneficial for autologous cell and gene therapies. This review focuses on the potential of adult stem cells to give rise to hematopoietic and vascular cells. We discuss evidence that a highly purified population of adult stem cells, termed SP cells, serves as a hematopoietic progenitor and can contribute to vascular regeneration after injury. We also discuss the potential relationship of these cells to the embryonic hemangioblast.     

Adult and embryonic blood and endothelium derive from distinct precursor populations which are differentially programmed by BMP in Xenopus

Blood and blood vessels develop in close association in vertebrate embryos and loss-of-function mutations suggest common genetic regulation. By the criteria of co-expression of blood and endothelial genes, and lineage tracing of progeny, we locate two distinct populations of progenitors for blood and endothelial cells in developing Xenopus embryos. The first population is located immediately posterior to the cement gland during neurula stages and gives rise to embryonic blood and vitelline veins in the anterior ventral blood island (aVBI), and to the endocardium of the heart. The second population resides in the dorsal lateral plate mesoderm, and contains precursors of adult blood stem cells and the major vessels. Both populations differentiate into endothelial cells in situ but migrate to new locations to differentiate into blood, suggesting that their micro-environments are unsuitable for haematopoietic differentiation. Both require BMP for their formation, even the Spemann organiser-derived aVBI, but individual genes are affected differentially. Thus, in the embryonic population, expression of the blood genes, SCL and GATA2, depend on BMP signalling while expression of the endothelial gene, Xfli1, does not. By contrast, Xfli1 expression in the adult, DLP population does require BMP. These results indicate that both adult and the anterior component of embryonic blood in Xenopus embryos derive from populations of progenitors that also give rise to endothelial cells. However, the two populations give rise to distinct regions of the vasculature and are programmed differentially by BMP.     

Calcium current in embryonic Xenopus muscle cells in culture

We have investigated the appearance of calcium current in Xenopus muscle cells in 1- to 6-day-old cultures. Whole cell currents were recorded using a patch-clamp amplifier with sodium and potassium replaced with tetraethylammonium and cesium, respectively, and BaCl2 used in place of CaCl2. When the muscle membrane was depolarized above -30 mV, a slow inward current was activated, the current reached a peak amplitude near 0 mV, and an outward current became apparent above +10 mV. This slow current was enhanced by adding barium or Bay K 8644 to the extracellular recording solution and was blocked by the addition of cobalt, cadmium, or the dihydropyridines nifedipine or (+)PN 200-110. Taken together these results indicate the presence of an inward calcium current mediated through L-type channels. Thirty-one percent of the cells examined on the first day in culture showed no discernible slow inward current; however, as the age of the culture increased, all cells showed slow inward current and there was an increase in the amplitude of the current. A small proportion of the muscle cells (5 out of 34) also showed a fast activating and inactivating inward current. This current, which activated at more hyperpolarized potentials (-40 mV) was only present when 5 mM ATP was included in the internal recording solution. It also appeared to be mediated through a calcium channel but not a dihydropyridine, sensitive channel.     

Genes and mechanisms involved in early embryonic development in Xenopus laevis

Our laboratory is studying genes involved in the regulation of the balance between cell growth and differentiation during embryonic development in Xenopus. We have analyzed the developmental expression of the proto-oncogenes c-myc, and KiRas 2B, the proliferating cell nuclear antigen (PCNA), and the tumor suppressor gene p53. These genes, usually expressed during cell proliferation, are expressed in the oocyte in large quantities, but the majority of their maternal RNAs are degraded by the gastrula stage. The expression of c-myc and the localization of the protein indicate that c-myc has the characteristics expected for a gene involved in the regulation of the mid-blastula transition, when zygotic expression is turned on in the embryo. Its expression during late development or during regeneration indicates that it enables the cells to remain competent for cycling during organogenesis. In vitro systems that reproduce the principal cellular functions during early development are used as model systems to understand the mechanisms involved in early embryogenesis.