Maxadilan Activates PAC1 Receptors Expressed in Xenopus laevis Melanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long‐term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non‐transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

非洲爪蟾眼的形态发生研究

详细观察和描述了非洲爪蟾Xenopus laevis眼的发生和发育变化过程,并分别对各发育时期视网膜的厚度进行了定量分析.非洲爪蟾眼的发牛开始于眼原基的形成,进而形成视泡;晶状体的发生是在视杯外壁增厚的同时诱导覆盖其上的胚胎外胚层内层增厚,形成预定晶状体板;在视网膜和晶状体共同诱导下,预定角膜上皮变为透明的角膜.在视杯出现之前,预定RPE的厚度由厚变薄,NR层不断地增厚直至结构功能完善.     

Expression of Size-Selected RNA Encoding Brain Serotonin Transporter in Xenopus laevis Oocytes

The mRNA that encodes a serotonin transporter was expressed using the Xenopus laevis oocyte expression system. Poly(A)+ RNA isolated from mouse brainstem was injected into Xenopus laevis oocytes, and the ability of oocytes to take up serotonin was measured 3 days postinjection. RNA-dependent serotonin uptake was sensitive to citalopram, a specific inhibitor of serotonin uptake, whereas background levels of serotonin uptake were not citalopram sensitive. Two RNA size fractions, 4.0 and 4.5 kb, were most efficient in stimulating uptake. Injection into Xenopus laevis oocytes of the 4.5-kb size fraction of mouse brainstem RNA resulted in threefold more serotonin uptake than did injection of unfractionated poly(A)+ RNA.     

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.     

Functional expression of basolateral Cl−/HCO3− exchange from rat jejunum in Xenopus laevis oocytes

Poly(A)⁺ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of Cl⁻/HCO₃⁻ antiport was investigated by means of ³⁶Cl⁻ uptake. Two days after injection of 50 ng of poly(A)⁺ RNA, Cl⁻ uptake was significantly increased with respect to water-injected oocytes. The expressed transport was inhibited by 0·2 mM DIDS, whereas endogenous Cl⁻ uptake was unaffected by this disulphonic stilbene. After sucrose density gradient fractionation, the highest expression of DIDS-sensitive Cl⁻ uptake was detected with mRNA size fraction of about 2–4 kb in length. The expressed Cl⁻ uptake can occur against a Cl⁻ concentration gradient and is unaffected by the known Cl⁻ channel blocker anthracene-9-carboxylic acid. Cl⁻ transport mechanism has properties similar to jejunal basolateral Cl⁻/HCO₃⁻ exchange with regard to Na⁺ dependence. © 1998 John Wiley & Sons, Ltd.     

Localization of cortical granule lectin ligand in Xenopus laevis egg jelly

The block to polyspermy in Xenopus laevis involves an interaction between a cortical granule lectin, released at fertilization, and a ligand located in the egg extracellular matrix. The egg extracellular matrix in X. laevis consists of a vitelline envelope and three distinct jelly layers, designated J1, J2 and J3. To localize cortical granule lectin ligand in the egg extracellular matrix, we used enzyme-linked lectin assays that showed that cortical granule lectin ligands were absent in J2, J3 and the vitelline envelope. Cortical granule lectin bound to a ligand(s) in J1 in a galactose-dependent fashion. In addition, we separated egg jelly macromolecules electrophoretically and, in conjunction with western blotting, have shown that J1 contains two major, high molecular weight ligands for cortical granule ligand. Finally, using confocal microscopy, we demonstrated that the ligand(s) for cortical granule lectin occupies a 20–30 μm thick band in a region of J1 just proximal to the vitelline envelope.     

Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF-2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)-expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all-trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.     

Cell cycle transition in early embryonic development of Xenopus laevis

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.     

Expression of Functional Bradykinin Receptors in Xenopus Oocytes

mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.     

Comparative molecular phylogeography of two Xenopus species, X. gilli and X. laevis, in the south-western Cape Province, South Africa

Xenopus gilli is a vulnerable anuran with a patchy distribution along the south-western coast of the Cape Province, South Africa. This species is sympatric with Xenopus laevis laevis , a widespread relative found over much of southern Africa. We examined the molecular phylogeography and population structure of the contact zone between these species to obtain information about historical biogeography and conservation management of this region. Analyses of the distribution, frequency, and cladistic and phenetic relationships among mitochondrial DNA haplotypes indicate that population subdivision is present in both taxa but that long-term isolation of sets of populations has occurred in X. gilli only. Haplotype and nucleotide diversity are also considerably higher within and among X. gilli ponds than X. l. laevis ponds in this region. We attribute the genetic segregation of X. gilli populations to ancient habitat fragmentation by ocean transgression into X. gilli habitat and to continued habitat alteration by human activity. The lower level of genetic diversity in X. l. laevis in this region is likely a result of a recent arrival of this taxon to the south-western Cape region relative to X. gilli . Population structure in X. l. laevis may be a result of isolation by distance. Clear evidence exists for at least two management units within X. gilli and strongly supports the establishment of protective measures east of False Bay in order to conserve a substantial portion of this species' extant genetic diversity.     

Changes in the adhesive properties of dissociated and reaggregated Xenopus laevis embryo cells

Activin A is a member of the transforming growth factor beta superfamily, and the strongest candidate mesoderm-inducer. The initial adhesive property changes in amphibians are likely to be mediated by mesoderm-inducers like activin A. The manner in which these changes actually occur, however, remains poorly understood. In the present study, the adhesive property changes mediated by activin A were directly demonstrated. Activin A functioned as a morphogen at low concentrations (less than 0.5 ng/mL), with no effect on the type A adhesive property. But at high concentrations (1 ng/mL), it induced another type of adhesive property, type N, and at very high concentrations (more than 10 ng/mL), it induced yet another type of adhesive property, type Y. Cells that have types A, N, and Y adhesive properties ultimately differentiated into atypical epidermis, notochord, and yolk-rich cells, respectively. It was also shown that these changes occurred between 5 and 10 h after induction by activin A. The implications of these results for the relationship between the adhesive property acquired during early and later stages of differentiation are also discussed.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

Tetracycline-regulated gene expression switch in Xenopus laevis

Xenopus is a well-characterized model system for the investigation of biological processes at the molecular, cellular, and developmental level. The successful application of a rapid and reliable method for transgenic approaches in Xenopus has led to renewed interest in this system. We have explored the applicability of tetracycline-regulated gene expression, first described by Gossen and Bujard in 1992, to the Xenopus system. By optimizing conditions, tetracycline repressor induced expression of a luciferase reporter gene was readily and reproducibly achieved in both the Xenopus oocyte and developing embryo. This high level of expression was effectively abrogated by addition of low levels of tetracycline. The significance of this newly defined system for studies of chromatin dynamics and developmental processes is discussed.     

ras-p21 Activates phospholipase D and A2, but not phospholipase C or PKC,in Xenopus laevis Oocytes

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42^MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.