Pyramidal cell-to-inhibitory cell spike transduction explicable by active dendritic conductances in inhibitory cell

In the guinea-pig hippocampal CA3 region, the synaptic connection from pyramidal neurons tostratum pyramidale inhibitory neurons is remarkable. Anatomically, the connection usually consists of a single release site on an interneuronal dendrite, sometimes 200 m or more from the soma. Nevertheless, the connection is physiologically powerful, in that a single presynaptic action potential can evoke, with probability 0.1 to 0.6, a postsynaptic action potential with latency 2 to 6 ms. We construct a model interneuron and show that the anatomical and physiological observations can be reconciled if the interneuron dendrites are electrically excitable. Excitable dendrites could also account for depolarization-induced amplification of the pyramidal cell-interneuron EPSP in the voltage range subthreshold for spike generation.     

Ear manipulations reveal a critical period for survival and dendritic development at the single‐cell level in Mauthner neurons

Second‐order sensory neurons are dependent on afferents from the sense organs during a critical period in development for their survival and differentiation. Past research has mostly focused on whole populations of neurons, hampering progress in understanding the mechanisms underlying these critical phases. To move toward a better understanding of the molecular and cellular basis of afferent‐dependent neuronal development, we developed a new model to study the effects of ear removal on a single identifiable cell in the hindbrain of a frog, the Mauthner cell. Ear extirpation at various stages of Xenopus laevis development defines a critical period of progressively‐reduced dependency of Mauthner cell survival/differentiation on the ear afferents. Furthermore, ear removal results in a progressively decreased reduction in the number of dendritic branches. Conversely, addition of an ear results in an increase in the number of dendritic branches. These results suggest that the duration of innervation and the number of inner ear afferents play a quantitative role in Mauthner cell survival/differentiation, including dendritic development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1339–1351, 2015     

Properties of mouse retinal ganglion cell dendritic growth during postnatal development

The property of dendritic growth dynamics during development is a subject of intense interest. Here, we investigated the dendritic motility of retinal ganglion cells (RGCs) during different developmental stages, using ex vivo mouse retina explant culture, Semliki Forest Virus transfection and time-lapse observations. The results illustrated that during development, the dendritic motility underwent a change from rapid growth to a relatively stable state, i.e., at P0 (day of birth), RGC dendrites were in a highly active state, whereas at postnatal 13 (P13) they were more stable, and at P3 and P8, the RGCs were in an intermediate state. At any given developmental stage, RGCs of different types displayed the same dendritic growth rate and extent. Since the mouse is the most popular mammalian model for genetic manipulation, this study provided a methodological foundation for further exploring the regulatory mechanisms of dendritic development.     

Sexual differentiation and hormonal regulation of the laryngeal synapse in Xenopus laevis

In Xenopus laevis frogs, sex differences in adult laryngeal synapses contribute to sex differences in vocal behavior. This study explores the development of sex differences in types of neuromuscular synapses and the development and hormone regulation of sex differences in transmitter release. Synapses in the juvenile larynx have characteristics not found in adults: juvenile muscle fibers can produce subthreshold or suprathreshold potentials in response to the same strength of nerve stimulation and can also produce multiple spikes to a single nerve stimulus. Juvenile laryngeal muscle also contains the same synapse types (I, II, and III) as are found in adult laryngeal muscle. The distribution of laryngeal synapse types in juveniles is less sexually dimorphic than the distribution in adults. Analysis of quantal content indicates that laryngeal synapses characteristically release low amounts of transmitter prior to sexual differentiation. Quantal content values from male and female juveniles are similar to values for adult males and are lower than values for adult females. When juveniles are gonadectomized and treated with exogenous estrogen, quantal content values increase significantly, suggesting that this hormone may increase transmitter release at laryngeal synapses during development. Gonadectomy alone does not affect quantal content of laryngeal synapses in either sex. Androgen treatment decreases quantal content in juvenile females but not males; the effect is opposite to and smaller than that of estrogen. Thus, muscle fiber responses to nerve stimulation and transmitter release are not sexually dimorphic in juvenile larynges. Transmitter release is strengthened, or feminized, by the administration of estradiol, an ovarian steroid hormone. © 1995 John Wiley & Sons, Inc.     

Xenopus as a model to study alternative splicing in vivo

An increasing number of genes are being identified for which the corresponding mRNAs contain different combinations of the encoded exons. This highly regulated exon choice, or alternative splicing, is often tissue-specific and potentially could differentially affect cellular functions. Alternative splicing is therefore not only a means to increase the coding capacity of the genome, but also to regulate gene expression during differentiation or development. To both evaluate the importance for cellular functions and define the regulatory pathways of alternative splicing, it is necessary to progress from the in vitro or ex vivo experimental models actually used towards in vivo whole-animal studies. We present here the amphibian, Xenopus, as an experimental model highly amenable for such studies. The various experimental approaches that can be used with Xenopus oocytes and embryos to characterize regulatory sequence elements and factors are presented and the advantages and drawbacks of these approaches are discussed. Finally, the real possibilities for large-scale identification of mRNAs containing alternatively spliced exons, the tissue-specific patterns of exon usage and the way in which these patterns are modified by perturbing the relative amount of splicing factors are discussed.     

Down syndrome cell adhesion molecule is important for early development in Xenopus tropicalis

The Down syndrome cell adhesion molecule (DSCAM) is an Ig containing cell adhesion molecule with remarkable structural conservation throughout metazoans. In insects, DSCAM has 38,000 potential isoforms that convey axon guidance, fasciculation, and dendrite morphogenesis during neurodevelopment. In vertebrates, DSCAM is expressed throughout the nervous system and seems to also mediate proper axonal guidance and synaptogenesis without the isoform diversity found in insects. Differences in DSCAM function among several vertebrate species complicate the understanding of an evolutionarily conserved role during embryogenesis. We take advantage of the frog developmental model Xenopus tropicalis to study DSCAM function in early development by expression analysis and morpholino‐mediated knockdown. Our results indicate that DSCAM is expressed early in development and restricted to the head and nervous system. Knockdown of protein expression results in early morphogenetic phenotypes characterized by failed gastrulation and improper posterior neural tube closure. Our results reveal a specific, fundamental role of DSCAM in early morphogenetic movements, presumably through its well‐known role in homophilic cell adhesion. genesis 52:849–857, 2014. © 2014 Wiley Periodicals, Inc.     

A calcium-binding motif in SPARC/osteonectin inhibits chordomesoderm cell migration during Xenopus laevis gastrulation: Evidence of counter-adhesive activity in vivo

Secreted protein, acidic, rich in cysteine (SPARC) is a Ca2+-binding, counter-adhesive, extracellular glycoprotein associated with major morphogenic events and tissue remodeling in vertebrates. In Xenopus laevis embryos, SPARC is expressed first by dorsal mesoderm cells at the end of gastrulation and undergoes complex, rapid changes in its pattern of expression during early organogenesis. Another study has reported that precocious expression of SPARC by injection of native protein into the blastocoele cavity of pregastrula embryos leads to a concentration-dependent reduction in anterior development. Thus, normal development requires that the timing, spatial distribution, and/or levels of SPARC be regulated precisely. In a previous study, we demonstrated that injection of a synthetic peptide corresponding to the C-terminal, Ca2+-binding, EF-hand domain of SPARC (peptide 4.2) mimicked the effects of native SPARC. In the present investigation, peptide 4.2 was used to examine the cellular and molecular bases of the phenotypes generated by the aberrant presence of SPARC. Exposure of late blastula embryos to LiCl also generated a concentration-dependent reduction in anterior development; therefore, injections of LiCl were carried out in parallel to highlight the unique effects of peptide 4.2 on early development. At concentrations that caused a similar loss in anterior development (60-100 ng peptide 4.2 or 0.25-0.4 microg LiCl), LiCl had a greater inhibitory effect on the initial rate of chordomesoderm cell involution, in comparison with peptide 4.2. However, as gastrulation progressed, peptide 4.2 had a greater inhibitory effect on prospective head mesoderm migration than that seen in the presence of LiCl. Moreover, peptide 4.2 and LiCl had distinct influences on the expression pattern of dorso-anterior markers at the neural and tail-bud stages of development. Scanning electron microscopy showed that peptide 4.2 inhibited spreading of migrating cells at the leading edge of the involuting chordomesoderm. While still in close proximity to the blastocoele roof, many of the cells appeared rounded and lacked lamellipodia and filopodia extended in the direction of migration. In contrast, LiCl had no effect on the spreading or shape of involuting cells. These data are the first evidence of a counter-adhesive activity for peptide 4.2 in vivo, an activity demonstrated for both native SPARC and peptide 4.2 in vitro.     

Hizikia fusiforme extract enhances dendritic cell maturation in vitro and in vivo

ABSTRACT

Dendritic cells (DCs) are play critical roles in the priming and regulation of immune responses. DCs rapidly process and convey these antigens to prime antigen-specific T cells. Therefore, regulation of DCs functions is important for immunity and immunotherapies. Immune adjuvants for DCs activation are needed to improve the efficacy of vaccines against tumors and many infectious diseases. Therefore, we demonstrate that H. fusiformis extract can regulate DCs maturation and activation. H. fusiformis extract induced costimulatory molecules (CD 80 and CD86), antigen-presenting molecules (major histocompatibility complex (MHC) I and II), CCR7 expression, and interleukin (IL)-12 production in DCs. These effects are associated with upregulation of mitogen-activated protein kinase (MAPK) signaling pathway. In addition, H. fusiformis extract induces costimulatory molecules on splenic DCs and activated CD8⁺ T cells in vivo. Taken together, these findings suggest that H. fusiformis extract may be a potential efficient immune therapeutic compound in DCs-mediated immunotherapies.     

Relationship between ectopic germinal vesicle breakdown in Xenopus oocytes and dorsal development of the embryo

The early development of several species involves the segregation of cytoplasmic components into different regions of the egg. In Xenopus zygotes, a 30° rotation displaces the central animal cytoplasm to the future dorsal side of the embryo. To elucidate the role of the central animal cytoplasm in dorsal determination, we induced germinal vesicle breakdown (GVBD) closer to the equator by cold/centrifugation treatment of oocytes. Centrifugation moved the germinal vesicle to the centripetal side; eggs with such displaced GVBD fertilized and began to develop normally. Dorsal embryonic structures tended to develop on the GVBD side of the egg, but displacement of the GVBD was insufficient to rescue dorsal structures in axis-deficient embryos. The labeling of yolk platelets of oocytes with Trypan Blue revealed similar cytoplasmic patterns in control and treated eggs. Furthermore, 67% of treated eggs had Danilchik's swirl, indicative of the dorsal side, on the GVBD side. In conclusion, both the swirl and dorsal development tend to occur on the GVBD side of cold/centrifuged eggs; however, displaced GVBD cannot by itself determine dorsality.     

In vivo magnetic resonance microscopy of differentiation in Xenopus laevis embryos from the first cleavage onwards

Differentiation inside a developing embryo can be observed by a variety of optical methods but hardly so in opaque organisms. Embryos of the frog Xenopus laevis--a popular model system--belong to the latter category and, for this reason, are predominantly being investigated by means of physical sectioning. Magnetic resonance imaging (MRI) is a noninvasive method independent of the optical opaqueness of the object. Starting out from clinical diagnostics, the technique has now developed into a branch of microscopy--MR microscopy--that provides spatial resolutions of tens of microns for small biological objects. Nondestructive three-dimensional images of various embryos have been obtained using this technique. They were, however, usually acquired by long scans of fixed embryos. Previously reported in vivo studies did not cover the very early embryonic stages, mainly for sensitivity reasons. Here, by applying high field MR microscopy to the X. laevis system, we achieved the temporal and spatial resolution required for observing subcellular dynamics during early cell divisions in vivo. We present image series of dividing cells and nuclei and of the whole embryonic development from the zygote onto the hatching of the tadpole. Additionally, biomechanical analyses from successive MR images are introduced. These results demonstrate that MR microscopy can provide unique contributions to investigations of differentiating cells and tissues in vivo.     

ccl19 and ccl21 affect cell movements and differentiation in early Xenopus development

We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal–ventral patterning during early embryogenesis in Xenopus.     

Fission yeast tmsl protein abrogates normal development in Xenopus laevis embryos

Recently we cloned tms1 (a putative dehydrogenase) by complementation of a human tumour-derived mutant p53 induced growth arrest in fission yeast. Microinjection of purified tmsl protein into Xenopus laevis embryos abrogated normal embryo development by causing cleavage retardation or cleavage arrest of injected blastomeres in a concentration dependant manner, whereas injection of specific affinity purified tms1 antiserum showed no significant morphological defects. Microinjection of tms1 protein together with affinity purified tms1 antibody resulted in a significantly reduced number of cleavage arrested embryos.     

Xenopus cyclin D2: Cloning and expression in oocytes and during early development

Summary— We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis cyclin D2 protein. Cyclin D2 mRNA is identified as a member of the class of maternal RNAs. It is rare and stable during embryonic development at least until tadepole. In addition, a second cDNA coding for a truneated version of cyclin D2 was also isolated. Mieroinjection of cyclin D2 into oocytes undergoing meiotic maturation and parthenogenetic activation reveals that the protein is stable for several hours, independently of the ubiquitin-mediated degradation of cyclin B2 that takes place periodically during this process. Microinjected cyclin D2 localizes both in the cytoplasm and in the nucleus of oocyte. In somatic cells, it is well established that cyclin D2 is almost exclusively nuclear and very labile. The unusual behaviour of cyclin D2 upon injection into oocytes may provide indications about a possible role for this protein during meiosis and early development.     

Magnetic resonance and near-infrared imaging using a novel dual-modality nano-probe for dendritic cell tracking in vivo

Background aimsThe effect of cellular-based immunotherapy is highly correlated with the success of dendritic cells (DCs) homing to the draining lymph nodes (LNs) and interacting with antigen-specific CD4⁺ T cells. In this study, a novel magneto-fluorescent nano-probe was used to track the in vivo migration of DCs to the draining LNs.MethodsA dual-modality nano-probe composed of superparamagnetic iron oxide (SPIO) and near-infrared fluorescent (NIRF) dye (NIR797) was developed, and its magnetic and optical contrasting properties were characterized. DCs generated from mouse bone marrow were co-cultured with the probe at a lower concentration of 10 μg/mL. The cell phenotype and function of DCs were also investigated by fluorescence-activated cell sorting analysis and mixed leukocyte reactivity assay. Labeled DCs were injected into the footpad of C57BL/6 mice. Afterward, magnetic resonance imaging, NIRF imaging, Perls staining and CD11c immunofluorescence were used to observe the migration of the labeled DCs into draining LNs.ResultsThe synthetic SPIO-NIR797 nano-probe had a desirable superparamagnetic and near-infrared behavior. Perls staining showed perfect labeling efficiency. The cell phenotypes, including CD11c, CD80, CD86 and major histocompatibility complex class II, as well as the T-cell activation potential of the mature DCs were insignificantly affected after incubation (P > 0.05). Labeled DCs migrating into LNs could be detected by both magnetic resonance imaging and NIRF imaging simultaneously, which was further confirmed by Perls staining and immunofluorescence.ConclusionsThe novel dual-modality SPIO-NIR797 nano-probe has highly biocompatible characteristics for labeling and tracking DCs, which can be used to evaluate cancer immunotherapy in clinical applications.     

Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones

Formation of neurites and their differentiation into axons and dendrites requires precisely controlled changes in the cytoskeleton. While small GTPases of the Rho family appear to be involved in this regulation, it is still unclear how Rho function affects axonal and dendritic growth during development. Using hippocampal neurones at defined states of differentiation, we have dissected the function of RhoA in axonal and dendritic growth. Expression of a dominant negative RhoA variant inhibited axonal growth, whereas dendritic growth was promoted. The opposite phenotype was observed when a constitutively active RhoA variant was expressed. Inactivation of Rho by C3-catalysed ADP-ribosylation using C3 isoforms (Clostridium limosum, C3(lim) or Staphylococcus aureus, C3(stau2)), diminished axonal branching. By contrast, extracellularly applied nanomolar concentrations of C3 from C. botulinum (C3(bot)) or enzymatically dead C3(bot) significantly increased axon growth and axon branching. Taken together, axonal development requires activation of RhoA, whereas dendritic development benefits from its inactivation. However, extracellular application of enzymatically active or dead C3(bot) exclusively promotes axonal growth and branching suggesting a novel neurotrophic function of C3 that is independent from its enzymatic activity.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.     

Repulsive guidance molecule A (RGM A) and its receptor neogenin during neural and neural crest cell development of Xenopus laevis

Background information. RGM A (repulsive guidance molecule A) is a GPI (glycosylphosphatidylinositol)‐anchored glycoprotein which has repulsive properties on axons due to the interaction with its receptor neogenin. In addition, RGM A has been demonstrated to function as a BMP (bone morphogenetic protein) co‐receptor. Results. In the present study, we provide the first analysis of early RGM A and neogenin expression and function in Xenopus laevis neural development. Tissue‐specific RGM A expression starts at stage 12.5 in the anterior neural plate. Loss‐of‐function analyses suggest a function of RGM A and neogenin in regulating anterior neural marker genes, as well as eye development and neural crest cell migration. Furthermore, overexpression of RGM A leads to ectopic expression of neural crest cell marker genes. Conclusions. These data indicate that RGM A and neogenin have important functions during early neural development, in addition to their role during axonal guidance and synapse formation.     

Changing dendritic field size of mouse retinal ganglion cells in early postnatal development

During early postnatal development, dendrites of retinal ganglion cells (RGCs) extend and branch in the inner plexiform layer to establish the adult level of stratification, pattern of branching, and coverage. Many studies have described the branching patterns, transient features, and regulatory factors of stratification of the RGCs. The rate of RGC dendritic field (DF) expansion relative to the growing retina has not been systematically investigated. In this study, we used two methods to examine the relative expansion of RGC DFs. First, we measured the size of RGC DFs and the diameters of the eyeballs at several postnatal stages. We compared the measurements with the RGC DF sizes calculated from difference of the eyeball sizes based on a linear expansion assumption. Second, we used the number of cholinergic amacrine cells (SACs) circumscribed by the DFs of RGCs at corresponding time points as an internal ruler to assess the size of DFs. We found most RGCs exhibit a phase of faster expansion relative to the retina between postnatal day 8 (P8) and P13, followed by a phase of retraction between P13 and adulthood. The morphological α cells showed the faster growing phase but not the retraction phase, whereas the morphological ON–OFF direction selective ganglion cells expanded in the same pace as the growing retina. These findings indicate different RGCs show different modes of growth, whereas most subtypes exhibit a fast expansion followed by a retraction phase to reach the adult size. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 397–407, 2010     

Fluorescent labeling of endothelial cells allows in vivo,continuous characterization of the vascular development of Xenopus laevis

Appropriate blood supply and vascular development are necessary in development and in cancer, heart disease, and diabetes. Here, we report the use of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) to label endothelial cells and characterize the vasculature of live Xenopus embryos. The atlas we have created provides a detailed map of normal vascular development against which perturbations of normal patterning can be compared. By following the development of the intersomitic vessels in real-time, we show that, while rostrocaudal gradient of maturing intersomitic vessels occurs, it is not absolute. In addition, the comparative study of the ontogeny of nerve bundles from the spinal cord of transgenic Xenopus embryos expressing green fluorescent protein in the nervous system and blood vessels demonstrates a strong anatomical correlation in neurovascular development. These studies provide the basis for understanding how the vascular system forms and assumes its complicated stereotypical pattern in normal development and in disease.     

Xenopus laevis tadpole limb regeneration in vivo and in vitro: thyroxine directly promotes blastemal cell proliferation and morphogenesis

Regeneration in hindlimbs of Xenopus laevis larvae which were amputated at stage 53 and 55 through the tarsalia region is promoted by thyroxine (T4), while propyl-thiouracil (PTU) inhibits regeneration when compared to controls. In this paper, by in vivo and in vitro experiments, we demonstrate that the promoting effect of T4 on the regenerative processes of larval X. laevis hindlimbs is a direct effect of this hormone on the blastemal cells. By contrast, the inhibitory effect of PTU on the regenerative process is not due to a direct effect on blastemal cells or to a general toxic effect on the treated larvae, but is related to hypothyroidism induced by the drug. We find that: (i) an increase in blastemal cell proliferation is observed not only in blastemata of T4-treated larvae, but also in blastemata cultured in vitro in a medium supplemented with T4; (ii) the renegerative process is accelerated not only in larvae reared in T4 but also in larvae submitted to a combined treatment of T4 and PTU; (iii) inhibition of cell proliferation is observed in blastemata of PTU-reared larvae but not in blastemata cultured in vitro in a medium supplemented with PTU. Experiments on thyroidless larvae (which were submitted to transplantation of hindlimbs from larvae at stages 53 and 55 followed by amputation of their own right hindlimb and the transplanted limbs) have shown that without thyroid hormone the regenerative process is arrested at cone stage and the promoting effect of T4 treatment is dependent on limb stage and amputation level.