Expression and role of Roundabout-1 in embryonic Xenopus forebrain.

The receptor Roundabout-1 (Robo1) and its ligand Slit are known to influence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. We report here the identification and expression of a Xenopus Robo1 orthologue that is highly homologous to mammalian Robo1. By using overexpression studies and immunohistochemical and in situ hybridization techniques, we have investigated the role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robo1 in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain.     

Co-expression in Xenopus neurons and neuroendocrine cells of messenger RNA homologues of exocytosis proteins DOC2 and munc18-1.

The proteins munc18-1 and DOC2 are assumed to play a role in docking of synaptic vesicles in neurotransmitter exocytosis at the presynaptic junction. As the proteins are known to interact, they should co-exist within neurons. We have tested this hypothesis for exocytosis of both classical and peptidergic messengers, by investigating the distribution of the messenger RNAs of munc 18-1 and DOC2 homologues in the brain and pituitary gland of the clawed toad Xenopus laevis, using in situ hybridization. For this purpose we cloned a partial complementary DNA encoding Xenopus unc18 (xunc18) and used a corresponding RNA probe, together with an RNA probe for Xenopus DOC2. At the messenger RNA level DOC2 and xunc18 were found to be expressed throughout the Xenopus brain. All brain nuclei expressing DOC2-messenger RNA showed xunc18-messenger RNA expression as well. Co-expression was shown at the individual cell level in consecutive sections of large-sized neurons. A strong expression was demonstrated in the suprachiasmatic and magnocellular nuclei and in peptidergic endocrine cells in the intermediate and anterior lobes of the pituitary gland, suggesting roles of DOC2 and xunc18 in messenger release from peptidergic secretory systems. Combined in situ hybridization and immunocytochemical analyses show that neuropeptide Y-containing cells in the suprachiasmatic nucleus also express DOC2 and xunc18 messenger RNAs. Since these cells have a high secretory activity, controlling the activity of the pituitary pars intermedia, the levels of expression of DOC2 and xunc18 may be indicators for neuronal secretory activity. The present data represent the first evidence for the co-existence of DOC2 and munc18-1 and suggest co-ordinate action of these proteins at the level of brain nuclei, individual neurons and endocrine cells.     

Selective regulation of xSlo splice variants during Xenopus embryogenesis

Calcium-activated potassium channels regulate excitability of the adult nervous system. In contrast, little is known about the contribution of calcium-activated potassium channels to excitability of the embryonic nervous system when electrical membrane properties and intracellular calcium levels show dramatic changes. Embryonic Xenopus spinal neurons exhibit a well-characterized developmental program of excitability that involves several different currents including calcium-activated ones. Here, we show that a molecular determinant of calcium-activated potassium channels, xSlo, is expressed during Xenopus embryogenesis even prior to differentiation of excitable tissues. Five different xSlo variants are expressed in embryonic tissues as a consequence of alternative exon usage at a single splice site. One of these variants, xSlo59, is neural-specific, and its expression is limited to late stages of neuronal differentiation. However, expression of the four other variants occurs in both muscle and neurons at all stages of development examined. Electrophysiological analysis of recombinant xSlo channels reveals that the xSlo59 exon serves as a gain-of-function module and allows physiologically relevant levels of membrane potential and intracellular calcium to activate effectively the resultant channel. These results suggest that xSlo59 channels play a unique role in sculpting the excitable membrane properties of Xenopus spinal neurons.     

NKCC1 (SLC12a2) induces a secondary axis in Xenopus laevis embryos independently of its co-transporter function

NKCC1 is a broadly expressed Na⁺–K⁺–Cl⁻ co-transporter involved in regulation of ion flux across the cell membrane and in regulating cell volume. Whilst much is known about the co-transporter activity of NKCC1 and its regulation by protein kinases and phosphatases, little is known about the activities of NKCC1 that are co-transporter independent. In this report we show that over-expression of NKCC1 in embryos of Xenopus laevis induces secondary axes , independently of its co-transporter activity. In addition, over-expression of NKCC1 results in the formation of neural tissue in ectodermal explants. We also show that NKCC1 is expressed broadly but non-uniformly in embryos of Xenopus laevis and Xenopus tropicalis , with prominent expression in the notochord, nervous system and stomach. These results provide insights into an additional, previously unreported activity of NKCCl.     

Thyroid hormone promotes neurogenesis in the Xenopus spinal cord.

Three phases of neurogenesis can be recognized during Xenopus spinal cord development. An early peak during gastrulation/neurulation is followed by a phase of low level neurogenesis throughout the remaining embryonic stages and a later peak at early larval stages. We show here that several genes known to be essential for early neurogenesis (X-NGNR-1, XNeuroD, XMyT1, X-Delta-1) are also expressed during later phases of neurogenesis in the spinal cord, suggesting that they are involved in regulating spinal neurogenesis at later stages. However, additional neuronal determination genes may be important during larval stages, because X-NGNR-1 shows only scant expression in the spinal cord during larval stages. Thyroid hormone treatment of early larvae promotes neurogenesis in the spinal cord, where thyroid hormone receptor xTRalpha is expressed from early larval stages onward and results in precocious up-regulation of XNeuroD, XMyT1, and N-Tubulin expression. Similarly, thyroid hormone treatments of Xenopus embryos, which were coinjected with xTRalpha and the retinoid X receptor xRXRalpha, repeatedly resulted in increased numbers of neurons, whereas unliganded receptors repressed neurogenesis. Our findings show that thyroid hormones are sufficient to up-regulate neurogenesis in the Xenopus spinal cord.     

Two Hoxc6 transcripts are differentially expressed and regulate primary neurogenesis in Xenopus laevis

Hox genes are key players in defining positional information along the main body axis of vertebrate embryos. In Xenopus laevis, Hoxc6 was the first homeobox gene isolated. It encodes two isoforms. We analyzed in detail their spatial and temporal expression pattern during early development. One major expression domain of both isoforms is the spinal cord portion of the neural tube. Within the spinal cord and its populations of primary neurons, Hox genes have been found to play a crucial role for defining positional information. Here we report that a loss‐of‐function of either one of the Hoxc6 products does not affect neural induction, the expression of general neural markers is not modified. However, Hoxc6 does widely affect the formation of primary neurons within the developing neural tissue. Manipulations of Hoxc6 expression severly changes the expression of the neuronal markers N‐tubulin and Islet‐1. Formation of primary neurons and formation of cranial nerves are affected. Hence, Hoxc6 functions are not restricted to the expected role in anterior‐posterior pattern formation, but they also regulate N‐tubulin, thereby having an effect on the initial formation of primary neurons in Xenopus laevis embryos. Developmental Dynamics 238:755–765, 2009. © 2009 Wiley‐Liss, Inc.     

Embryonic expression and extracellular secretion of Xenopus slit

The slit genes have recently been found to encode proteins with a conserved chemorepulsive activity for axons in invertebrates and vertebrates. We have determined the expression pattern of a slit gene in Xenopus embryos. In the neural tube, slit is expressed at the ventral and dorsal midlines, and the motor neurons. slit is also expressed in a changing pattern in the retina. The full-length Xenopus Slit protein is secreted extracellularly, whereas its receptor Roundabout can not be secreted. Using a myc-tagged secreted Slit protein, we confirmed the binding of Slit to Roundabout expressed on the cell surface.These results confirm Slit-Roundabout interactions and the biochemical properties of Slit and Roundabout proteins, and further support the idea that Slit may guide axon projections in multiple regions of the embryo.     

Characterization of olfactory receptor organs in Xenopus laevis Daudin.

Xenopus laevis is highly suitable for studying the mechanisms of olfactory reception for water-soluble odorants and for airborne odorants. However, the functional differences of cells and component protein molecules in the olfactory receptors of Xenopus have remained obscure. In recent studies, the patterns of sugar residues expressed on the cell surface have been utilized to analyze the characteristics of neurons, because the sugar chains in neurons play very important roles in targeting and cell-to-cell communication. In this study, we have determined the distribution of sugar residues and glycoproteins in the olfactory receptor organs of Xenopus using lectins as labeling agents, and characterized the receptors of water-soluble odorants and of airborne odorants. The results of lectin histochemical analysis show distributional differences of GlcNAc, GalNAc and mannose between the middle chamber and the lateral chamber of the main nasal cavity. Furthermore, a 65 kDa glycoprotein containing mannose, GlcNAc and GalNAc was specifically detected in the medial chamber of the main cavity epithelium in receptor organs of airborne odorants by SDS-PAGE and lectin blotting. The characteristics of the epithelia demonstrated in this study should further our understanding of the functional differences between the receptors of water-soluble odorants and of airborne odorants at the molecular level.     

A cell surface marker of thymus-dependent lymphocytes in Xenopus laevis is identifiable by mouse monoclonal antibody

A mouse monoclonal antibody (mAb), named XT-1 (IgG2b,lambda), was prepared against J strain Xenopus laevis thymocytes. The determinant recognized by this mAb (XT-1 determinant) was expressed by 92-98% thymocytes and 22-37% splenic lymphocytes from intact J strain frogs and larvae, as analyzed by flow cytofluorometry. The XT-1 determinant was also expressed by 20-30% peripheral blood lymphocytes and 5-11% bone marrow lymphocytes from adult, and 11-17% liver lymphocytes from larval J strain Xenopus. Early larval thymectomy depleted XT-1-positive cells in the spleen to less than 2% of the total lymphocyte population. Double immunofluorescence experiments revealed that XT-1+ lymphocytes in the spleen are surface IgM-. The strain distribution study on the expression of XT-1 determinant suggests the allelic polymorphism of the XT-1 determinant. The surface antigen identified by the present XT-1 mAb may provide an important marker for lymphocytes in thymus-dependent lineage in Xenopus.     

Observations on the development of cerebellar afferents in Xenopus laevis

Summary The development of cerebellar afferents has been studied in the clawed toad, Xenopus laevis, from stage 46 to 64, with the horseradish peroxidase retrograde tracer technique. Already in stage 48 tadpoles, i.e. before the formation of the limbs, a distinct set of cerebellar afferents was found. Vestibulocerebellar (mainly arising bilaterally in the nucleus vestibularis caudalis) and contralateral olivocerebellar projections dominate. Secondary trigeminocerebellar (from the descending nucleus of the trigeminal nerve) and reticulocerebellar connections were also found. At stage 50, spinocerebellar projections appear originating from cervical and lower thoracic/upper lumbar levels. The cells of origin of the spinocerebellar projection can be roughly divided in two neuronal types: ipsilaterally projecting large cells, which show a marked resemblance to primary motoneurones (spinal border cells) and smaller contralaterally projecting neurons. Primary spinocerebellar projections from spinal ganglion cells could not be demonstrated.At stage 50, a possible anuran homologue of the mammalian nucleus prepositus hypoglossi was found to project to the cerebellum. In only one of the experiments labeled neurons were found in the contralateral mesencephalic tegmentum. At none of the studied stages a raphecerebellar projection could be demonstrated.It appears that already early in cerebellar development, before the formation of the limbs, most of the cerebellar afferents as found in adult Xenopus laevis are present.     

Comparative analysis of embryonic gene expression defines potential interaction sites for Xenopus EphB4 receptors with ephrin-B ligands.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, act as signaling molecules regulating the migratory behavior of neurons and neural crest cells, and are implicated in tissue patterning, blood vessel formation, and tumorigenesis. On the basis of structural similarities and overlapping binding specificities, Eph receptors as well as their ligands can be divided into A and B subfamilies with orthologues found in all vertebrates. We describe here the isolation of cDNAs encoding Xenopus EphB4 receptors and show that embryonic expression is prominently associated with the developing vasculature, newly forming somites, the visceral arches, and non-neuronal tissues of the embryonic head. In a screen to identify potential ligands for EphB4 in Xenopus embryos, we isolated cDNAs for the Xenopus ephrin-B2 and -B3, which demonstrates that the Xenopus genome harbors genes encoding orthologues to all three currently known mammalian ephrin-B genes. We next performed in situ hybridizations to identify tissues and organs where EphB4 receptors may encounter ephrin-B ligands during embryonic development. Our analysis revealed distinct, but overlapping patterns of ephrin-B gene expression. Interestingly, each ephrin-B ligand displayed expression domains either adjacent to or within EphB4-expressing tissues. These findings indicate that EphB4 receptors may interact in vivo with multiple B-class ephrins. The expression patterns also suggest that EphB4 receptors and their ligands may be involved in visceral arch formation, somitogenesis, and blood vessel development.     

Noradrenergic excitation of a subpopulation of GABAergic cells in the basolateral amygdala via both activation of nonselective cationic conductance and suppression of resting K+ conductance: a study using glutamate decarboxylase 67-green fluorescent protein knock-in mice

GABAergic interneurons play central roles in the regulation of neuronal activity in the basolateral nucleus of the amygdala (BLA). They are also suggested to be the principal targets of the brainstem noradrenergic afferents which are involved in the enhancement of the BLA-related memory. In addition, behavioral stress has been shown to impair noradrenergic facilitation of GABAergic transmission. However, the noradrenaline (NA) effects in the BLA have not been differentiated among medium- to large-sized GABAergic neurons and principal cells, and remain to be elucidated in terms of their underlying mechanisms. Glutamate decarboxylase 67 (GAD67) is a biosynthetic enzyme of GABA and is specifically expressed in GABAergic neurons. To facilitate the study of the NA effects on GABAergic neurons in live preparations, we generated GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the control of an endogenous GAD67 gene promoter. Here, we show that GFP was specifically expressed in GABAergic neurons in the BLA of this GAD67-GFP knock-in mouse. Under whole-cell patch-clamp recordings in vitro, we identified a certain subpopulation of GABAergic neurons in the BLA chiefly on the basis of the electrophysiological properties. When depolarized by a current injection, these neurons, which are referred to as type A, generated action potentials at relatively low frequency. We found that NA directly excited type-A cells via alpha1-adrenoceptors, whereas its effects on the other types of neurons were negligible. Two ionic mechanisms were involved in this excitability: the activation of nonselective cationic conductance and the suppression of the resting K+ conductance. NA also increased the frequency of spontaneous IPSCs in the principal cells of the BLA. It is suggested that the NA-dependent excitation of type-A cells attenuates the BLA output for a certain period.     

Expression of synaptic vesicle two-related protein SVOP in the developing nervous system of Xenopus laevis.

Synaptic vesicle-associated proteins are important regulators of neurotransmitter release at synaptic terminals in mature animals. Some synaptic vesicle-associated proteins are also expressed during development, although their contribution to development is not as clear. Here, we describe the cloning and developmental expression pattern of the Xenopus laevis synaptic vesicle-associated protein SVOP, a gene first identified as an immediate target for proneural basic helix-loop-helix factors. Alignment analysis revealed a high level of identity between the SVOP protein sequences from Xenopus and other vertebrates. In developing Xenopus embryos, SVOP expression is restricted to the nervous system and is first detectable at the mid-neurula stage. As development progresses SVOP becomes broadly expressed throughout the central nervous system. Our observation that SVOP is expressed in the developing Xenopus nervous system suggests that it may be involved in neuron formation, maturation, or neuronal function.