Dissection,Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation^1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates ^12,14-18.While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells ^7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues ^{8,19-22,24-33}. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level ^{5,8,21,24,27-30,33-39}. Xenopus laevis, a classic model system for the study of early neural development ^{19,27,29,31-32,40-42}, serves as a particularly suitable system for retinal primary cell culture ^10,38,43-45.Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction ^25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products ^10,24,44-45.However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).     

Embryonic wound healing by apical contraction and ingression in Xenopus laevis

We have characterized excisional wounds in the animal cap of early embryos of the frog Xenopus laevis and found that these wounds close accompanied by three distinct processes: (1) the assembly of an actin purse-string in the epithelial cells at the wound margin, (2) contraction and ingression of exposed deep cells, and (3) protrusive activity of epithelial cells at the margin. Microsurgical manipulation allowing fine control over the area and depth of the wound combined with videomicroscopy and confocal analysis enabled us to describe the kinematics and challenge the mechanics of the closing wound. Full closure typically occurs only when the deep, mesenchymal cell-layer of the ectoderm is left intact; in contrast, when deep cells are removed along with the superficial, epithelial cell-layer of the ectoderm, wounds do not close. Actin localizes to the superficial epithelial cell-layer at the wound margin immediately after wounding and forms a contiguous "purse-string" in those cells within 15 min. However, manipulation and closure kinematics of shaped wounds and microsurgical cuts made through the purse-string rule out a major force-generating role for the purse-string. Further analysis of the cell behaviors within the wound show that deep, mesenchymal cells contract their apical surfaces and ingress from the exposed surface. High resolution time-lapse sequences of cells at the leading edge of the wound show that these cells undergo protrusive activity only during the final phases of wound closure as the ectoderm reseals. We propose that assembly of the actin purse-string works to organize and maintain the epithelial sheet at the wound margin, that contraction and ingression of deep cells pulls the wound margins together, and that protrusive activity of epithelial cells at the wound margin reseals the ectoderm and re-establishes tissue integrity during wound healing in the Xenopus embryonic ectoderm.     

Hypotonic Regulation of Mouse Epithelial Sodium Channel in Xenopus laevis Oocytes

The regulation of the epithelial Na⁺ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occur, i.e., migration, proliferation and cell absorption. Its sensitivity to hypotonically induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC. We used voltage-clamp techniques to study the amiloride-sensitive Na⁺ currents (INa_(amil)) and video microscopic methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly over 1.5 min. In contrast, there was no change in current amplitude of H₂O-injected oocytes to the osmotic insult. INa_(amil) kinetics analysis revealed a decrease in the slower inactivation time constant during the hypotonic stimuli. Currents from ENaC-injected oocytes were not sensitive to external Cl^? reduction. Neither short- nor long-term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 had reduced INa_(amil) hypotonic response compared to oocytes injected with wild-type ENaC subunits. Finally, during the hypotonic response ENaC-injected oocytes did not show a cell volume difference compared with water-injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane.     

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins ^1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references ^{1-6,7 ,8}).     

Imaging adhesion and signaling dynamics in Xenopus laevis growth cones

Xenopus laevis provides a robust model system to study cellular signaling and downstream processes during development both in vitro and in vivo. Intracellular signals must function within highly restricted spatial and temporal domains to activate specific downstream targets and cellular processes. Combining the versatility of developing Xenopus neurons with advances in fluorescent protein biosensors and imaging technologies has allowed many dynamic cellular processes to be visualized. This review will focus on the techniques we use to visualize and measure cell signaling, motility and adhesion by quantitative fluorescence microscopy in vitro and in vivo.     

A Novel Monoclonal Antibody disrupting Cell Type Specific Substratum Adhesion of Frog (Xenopus laevis) Epithelial Cells and Endothelial Cells

We isolated a mouse monoclonal antibody (FAD-II) that disrupts cell-substratum adhesion of amphibian ( Xenopus laevis ) epithelial cells and endothelial cells. The effect of the antibody was cell-type specific, and the antibody had no effect on fibroblastic cells while fibronectin peptide blocked cell-substratum adhesion of all the cell types examined. In developing frog embryos, the epitopes recognized by the antibody were detected in pronephrotic ducts and in other tissue cells of embryos (from stage 33/34 afterwards). In adult tissues, the antibody mainly recognized antigens in extracelluar matrices. The antigens recognized by the antibody seems to be novel glycoepitopes in frog cells.     

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.     

Quantitative changes in protein synthesis during oogenesis in Xenopus laevis

Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr⁻¹ while stage 6 oocytes make protein at a rate of 22.8 ng hr⁻¹. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile.     

Characterization of Single Inward Rectifier Potassium Channels from Embryonic Xenopus laevis Myocytes

Single inward rectifier K⁺ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K⁺ channels (Kir_(0.3), Kir_(0.5) and Kir_(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K⁺] _o respectively. Extracellular Ba²⁺ blocked the normally high channel activity in a concentration-dependent manner (K _A = 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K⁺ current, marked voltage dependence of Ba²⁺ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to E _K , outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg²⁺-free solution at pH 9.1. This was probably also true for Kir_(0.3), Kir_(0.5) and Kir_(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K⁺ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states. Received: 16 September 1996/Revised: 14 March 1997     

Protamine polymorphism in Xenopus laevis laevis

Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.     

利用修饰的随机引物构建非洲爪蟾酵母双杂交定向cDNA文库

描述了一种新的构建cDNA文库的方法,其中用来合成cDNA第一链的随机引物5'-端被加上碱基d(AC),在cDNA双链合成后所添加的连接头的末端含有碱基d(GTCG).在cDNA舣链3'-端,连接头连接上后会形成一个完整的Sall酶切位点d(GTCGAC),而在5'-端几乎不会形成Sall位点.在Sall酶切后利用形成的3'-粘性末端与5'-EcoRI粘性末端一起将cDNA双链定向导入线性化的质粒载体,再通过转化细菌获得cDNA文库.利用此方法构建了一个不同发育时期非洲爪蟾胚胎酵母双杂交cDNA文库.检测了文库中空载体的比例,插入片段大小和不同基因的表达水平几个指标,都符合预期,但是同时发现定位于mRNA的3'-端的插入片段比例比较低.这种倾向性符合文献报道,应该是实验系统的倾向性,不影响进一步的酵母双杂交实验.这些数据证明成功地构建了定向非洲爪蟾酵母双杂交cDNA文库.     

Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development

Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.     

Use of Hybrids between Xenopus laevis and Xenopus borealis in Chimera Formation: Dorsalization of Ventral Cells

The combination of Xenopus borealis and X. laevis provides an excellent cell marking system. The potential availability of this system for chimera formation has also been suggested. However, eggs and early embryos of these species differ in size and the fusion of blastomeres of different sizez results in some disturbance in arrangement of blastomeres of a chimera. This disturbance was avoided by use of embryos from X. laevis eggs fertilized with X. borealis sperm, instead of X. borealis embryos. The cells of these hybrids could also be distinguished from the cells of X. laevis.
The fate of animal ventral cells placed in the dorsal region was followed by making a chimera by fusing a right lateral half of an 8-cell X. laevis embryo with that of an 8-cell hybrid embryo. The animal ventral cells in the "dorsal" region were found to become "dorsalized", giving rise to a lateral half of dorsal axial structures. This observation explains a previous finding that the replacement of dorsal cells by ventral ones had no effect on embryogenesis in a composite embryo.     

Thyroid Hormone-Regulated Wnt5a/Ror2 Signaling Is Essential for Dedifferentiation of Larval Epithelial Cells into Adult Stem Cells in the Xenopus laevis Intestine

Background and Aims

Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis.

Methods/Findings

Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation.

Significance

Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.