Mutual inactivation of Notch receptors and ligands facilitates developmental patterning

Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.     

Expression of the NGF receptor gene in sensory neurons and their cutaneous targets prior to and during innervation

We have studied the expression of NGF receptor (NGFR) mRNA in the mouse trigeminal system at closely staged intervals throughout development. The level of NGFR mRNA per neuron is at a constant low level before the earliest axons reach the target field and increases 5-fold to plateau shortly after the arrival of the last axons. NGFR mRNA expression in developing target skin is restricted to mesenchyme, precedes the arrival of the earliest axons, and increases throughout the phase of target field innervation. These findings suggest that NGFR expression on sensory neurons occurs at a low level prior to target field innervation and is up-regulated with this event, and they raise the possibility that NGFRs on mesenchymal cells restrict the distribution of NGF in the target field.     

Lens-derived Semaphorin3A regulates sensory innervation of the cornea

The cornea, one of the most highly innervated tissues of the body, is innervated by trigeminal sensory afferents. During development, axons are initially repelled at the corneal margin, resulting in the formation of a circumferential nerve ring. The nature and source of guidance molecules that regulate this process remain a mystery. Here, we show that the lens, which immediately underlies the cornea, repels trigeminal axons in vivo and in vitro. Lens ablation results in premature, disorganized corneal innervation and disruption of the nerve ring and ventral plexus. We show that Semaphorin3A (Sema3A) is expressed in the lens epithelium and its receptor Neuropilin-1 (Npn1) is expressed in the trigeminal ganglion during cornea development. Inhibition of Sema3A signaling abrogates axon repulsion by the lens and cornea in vitro and phenocopies lens removal in vivo. These results demonstrate that lens-derived Sema3A mediates initial repulsion of trigeminal sensory axons from the cornea and is necessary for the proper formation of the nerve ring and positioning of the ventral plexus in the choroid fissure.     

Conformational changes in the negative arm of the circadian clock correlate with dynamic interactomes involved in post-transcriptional regulation

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Structure of the sensory innervation of the anal canal in the pig. A light- and electron-microscopical study

The sensory innervation of the anal canal of the pig was investigated by light and electron microscopy. The distribution of the different types of sensory nerve endings correlates with the histology of different zones: (1) After the rectal mucosa there was a zone lined with nonkeratinized stratified squamous epithelium. (2) A middle zone was lined with keratinized stratified squamous epithelium. Here the dermis already showed a papillary and reticular layer. (3) The last zone showed hairy skin with a high hair density. The following nerve endings were found: Free nerve endings reached the stratum superficiale in nonkeratinized squamous epithelium and the stratum granulosum in the keratinized squamous epithelium. Dermal free nerve endings were found in all zones near the epithelium and two different types were identified as those derived from C-fibers and those from A-delta-fibers. Merkel nerve endings showed different features depending on their location. Few Merkel-like cells were found in the epithelium of the anal crypts. Typical Merkel Tastscheiben were located at the base of epithelial ridges or pegs in zones 2 and 3. The number of Merkel cells varied up to 200. The myelinated afferent fiber supplied 10-15 Merkel cells. Merkel cells were also found regularly in the outermost layer of the external rooth sheath of hair follicles at about the same level as perifollicular nerve endings. Lamellated corpuscles were found in the dermis of all zones except the cranial part of zone 1, where the anal crypts are located. Generally they consisted of a central nerve terminal which may be branched. Each terminal was surrounded by an inner core of concentrically arranged lamellae of the terminal Schwann cell and one or several inner cores were included in a capsule of perineural cells. The size of the corpuscle, the regularity of the inner core and the number of capsular layers depended on the location of the corpuscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal development of sympathetic and sensory innervation of the rhesus monkey ovary.

We have used immunofluorescence to study the postnatal development of the sympathetic and sensory innervation to the rhesus monkey (Macaca mulatta) ovary. Sympathetic nerves were identified as adrenergic by their content of tyrosine hydroxylase (TH)-like immunoreactivity and as peptidergic by the presence of neuropeptide Y (NPY). Fibers containing substance P (SP) or calcitonin gene-related peptide (CGRP)-like immunoreactivity were considered as sensory, whereas vasoactive intestinal peptide (VIP)-positive fibers were only defined as peptidergic because VIP may be present in both sympathetic and sensory nerves. Ovaries from neonatal (2-mo-old), juvenile (9-18-mo-old), peripubertal (3-3.5-yr-old), adult (9-14-yr-old), and senescent (20-27-yr-old) monkeys were studied. At all ages, with the exception of senescence, TH-, NPY-, and VIP-containing fibers were associated with follicles in different developmental stages. In peripubertal and adult animals, some primordial follicles were found to be selectively innervated by VIPergic fibers that almost completely encircled each follicle. Both sympathetic and VIP fibers were also detected in the interstitial tissue and associated with the ovarian vasculature at all ages. The number of sympathetic and VIP fibers increased significantly (p < 0.01) between 2 mo and 9-18 mo of age, and again increased (p < 0.01) around the age of puberty (approximately 3 yr of age). After this time, the number of NPY and TH fibers remained constant. Conversely, the number of VIP fibers decreased (p < 0.05) by 9-14 yr of age, but remained constant thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Static and dynamic activity of cutaneous cold receptors induced by noradrenaline infusion

Experiments on rats showed that cutaneous cold receptors are sensitive to altered blood level of noradrenaline (NA). Both rise in and re-establishing of NA at a new, higher level exert an effect on firing activity in cutaneous cold receptors. When sustained at the raised level, NA alters both static and dynamic activity of cold receptors. The pattern of change (whether excitatory or inhibitory) depends on initial type of receptor activity.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 69–75, January–February, 1990.     

A rapid quantitative bioassay for evaluating the effects of ligands upon receptors that modulate cAMP levels in a melanophore cell line.

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the beta adrenergic receptor (beta AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

Molecular regulation of cognitive functions and developmental plasticity: impact of GABAA receptors

By controlling spike timing and sculpting neuronal rhythms, inhibitory interneurons play a key role in regulating neuronal circuits and behavior. The pronounced diversity of GABAergic (gamma-aminobutyric acid) interneurons is paralleled by an extensive diversity of GABAA receptor subtypes. The region- and domain-specific location of these receptor subtypes offers the opportunity to gain functional insights into the role of defined neuronal circuits. These developments are reviewed with regard to the regulation of sleep, anxiety, memory, sensorimotor processing and post-natal developmental plasticity.     

Biopanning and rapid analysis of selective interactive ligands.

Here we introduce a new approach for the screening, selection and sorting of cell-surface-binding peptides from phage libraries. Biopanning and rapid analysis of selective interactive ligands (termed BRASIL) is based on differential centrifugation in which a cell suspension incubated with phage in an aqueous upper phase is centrifuged through a non-miscible organic lower phase. This single-step organic phase separation is faster, more sensitive and more specific than current methods that rely on washing steps or limiting dilution. As a proof-of-principle, we screened human endothelial cells stimulated with vascular endothelial growth factor (VEGF) and constructed a peptide-based ligand-receptor map of the VEGF family. Next, we validated the motif PQPRPL as a novel chimeric ligand mimic that binds specifically to VEGF receptor-1 and to neuropilin-1. BRASIL may prove itself a superior method for probing target cell surfaces with a broad range of potential applications.     

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.     

The innervation pattern of the human Achilles tendon: studies of the normal and tendinosis tendon with markers for general and sensory innervation

Pain-free normal Achilles tendons and chronic painful Achilles tendons were examined by the use of antibodies against a general nerve marker (protein gene-product 9.5, PGP9.5), sensory markers (substance P, SP; calcitonin gene-related peptide, CGRP), and immunohistochemistry. In the normal tendons, immunoreactions against PGP9.5 and against SP/CGRP were encountered in the paratendinous loose connective tissue, being confined to nerve fascicles and to nerve fibers located in the vicinity of blood vessels. To some extent, these immunoreactions also occurred in the tendon tissue proper. Immunoreaction against PGP9.5 and against SP/CGRP was also observed in the tendinosis samples and included immunoreactive nerve fibers that were intimately associated with small blood vessels. In conclusion, Mechanoreceptors (sensory corpuscles) were occasionally observed, nerve-related components are present in association with blood vessels in both the normal and the tendinosis tendon.     

Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF receptor mRNAs deifferentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75^NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75^NGFR and trkA mRNA, may alter the ratio fo the two NGF receptors, and, conseuqnetly, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintenance of neural funciton. 1994 John Wiley & Sons, Inc.     

Expression of notch receptors and ligands in the adult gut.

The Notch signaling pathway has become recognized as a vitally important pathway in regulating proliferative/differentiative decisions and cell fate. To explore the involvement of the Notch pathway in adult gut, we investigated the expression of Notch receptors and their ligands by Northern blotting and in situ hybridization. Notch receptors and ligands were expressed in both proliferative and post-mitotic cells throughout adult rat gut, variously in epithelial, immune, and endothelial cells. Expression of Notch1, Jagged1, and Jagged2 frequently overlapped, whereas Notch2 expression was restricted to specific crypt cells, the lamina propria of the large intestine, and Peyer's patch lymphocytes. We propose that the expression of multiple Notch receptors and ligands in a range of different intestinal cell types indicates that this signaling pathway underpins many of the processes involved in the maintenance and function of the adult gut.