Lateral Signaling Research Articles

The Development of Archosaurian First-Generation Teeth in a Chicken Mutant

Modern birds do not have teeth. Rather, they develop a specialized keratinized structure, called the rhamphotheca, that covers the mandible, maxillae, and premaxillae. Although recombination studies have shown that the avian epidermis can respond to tooth-inductive cues from mouse or lizard oral mesenchyme and participate in tooth formation, attempts to initiate tooth development de novo in birds have failed. Here, we describe the formation of teeth in the talpid2 chicken mutant, including the developmental processes and early molecular changes associated with the formation of teeth. Additionally, we show recapitulation of the early events seen in talpid2 after in vivo activation of beta-catenin in wild-type embryos. We compare the formation of teeth in the talpid2 mutant with that in the alligator and show the formation of decidedly archosaurian (crocodilian) first-generation teeth in an avian embryo. The formation of teeth in the mutant is coupled with alterations in the specification of the oral/aboral boundary of the jaw. We propose an epigenetic model of the developmental modification of dentition in avian evolution; in this model, changes in the relative position of a lateral signaling center over competent odontogenic mesenchyme led to loss of teeth in avians while maintaining tooth developmental potential.

Intercellular coupling amplifies fate segregation during Caenorhabditis elegans vulval development

During vulval development in Caenorhabditis elegans, six precursor cells acquire a spatial pattern of distinct cell fates. This process is guided by a gradient in the soluble factor, LIN-3, and by direct interactions between neighboring cells mediated by the Notch-like receptor, LIN-12. Genetic evidence has revealed that these two extracellular signals are coupled: lateral cell-cell interactions inhibit LIN-3-mediated signaling, whereas LIN-3 regulates the extent of lateral signaling. To elucidate the quantitative implications of this coupled network topology for cell patterning during vulval development, we developed a mathematical model of LIN-3/LIN-12-mediated signaling in the vulval precursor cell array. Our analysis reveals that coupling LIN-3 and LIN-12 amplifies cellular perception of the LIN-3 gradient and polarizes lateral signaling, both of which enhance fate segregation beyond that achievable by an uncoupled system.

Expression of early placenta insulin-like growth factor in breast cancer cells provides an autocrine loop that predominantly enhances invasiveness and motility

Early placenta insulin-like growth factor (EPIL) is expressed by a subpopulation of the Her2-positive SKBR3 breast cancer cell line displaying high motility and transendothelial invasiveness in vitro, as recently shown by our group. As a consequence of this, we established cellular models by generating an EPIL-overexpressing SKBR3 cell line, knocked down EPIL by adding specific small interfering RNA (siRNA) to those cells and produced EPIL-enriched and depleted serum-free culture media. EPIL-expressing cells as well as EPIL-induced SKBR3 cells acquired a high capacity for transendothelial invasiveness. We observed a thin and outspread morphology caused by enhanced formation of lamellipodia, i.e. protrusions in the initial phase of motility. In parallel, Her2-positive MDAHer2 breast cancer cells also showed increased invasiveness when induced by EPIL-conditioned medium. A downstream signaling impact of EPIL could be observed in the form of reduced phosphorylation of Her2, erk1/2 and akt, while phospholipase Cgamma1 phophorylation remained unaffected. As an in vivo model for highly motile tumor cells, Paget's disease of the nipple showed simultaneous EPIL and Her2 expression upon immunohistochemical examination using specific antibodies. Such experimental data have been translated to a clinical setting by using a prognostic tissue microarray established from 603 breast cancer cases. Survival data analysis found a significant association between expression levels of EPIL and 5-year overall survival that was dose dependent: EPIL (negative) 84%, EPIL (moderately positive) 77%, EPIL (strongly positive) 48% (P < 0.005). One particular subgroup (7.6% of the cases with full clinical records) that comprised tumors simultaneously expressing EPIL and Her2 represented patients with the poorest 5-year overall survival. The results suggested that EPIL might be a cancer cell-produced growth factor that influences lateral Her2 signaling. Moreover, EPIL may be induced by factors apart from Her2 and may independently provide signaling for cancer invasion and motility.

Evolution and diversification of a sexually dimorphic luminescent system in ponyfishes (Teleostei: Leiognathidae), including diagnoses for two new genera.

A phylogeny was generated for Leiognathidae, an assemblage of bioluminescent, Indo-Pacific schooling fishes, using 6175 characters derived from seven mitochondrial genes (16S, COI, ND4, ND5, tRNA-His, tRNA-Ser, tRNA-Leu), two nuclear genes (28S, histone H3), and 15 morphological transformations corresponding to features of the fishes' sexually dimorphic light-organ system (LOS; e.g., circumesophageal light organ, lateral lining of the gas bladder, transparent flank and opercular patches). Leiognathidae comprises three genera, Gazza, Leiognathus, and Secutor. Our results demonstrate that Leiognathidae, Gazza, and Secutor are monophyletic, whereas Leiognathus is not. The recovered pattern of relationships reveals that a structurally complex, strongly sexually dimorphic and highly variable species-specific light organ is derived from a comparatively simple non-dimorphic structure, and that evolution of other sexually dimorphic internal and external features of the male LOS are closely linked with these light-organ modifications. Our results demonstrate the utility of LOS features, both for recovering phylogeny and resolving taxonomic issues in a clade whose members otherwise exhibit little morphological variation. We diagnose two new leiognathid genera, Photopectoralis and Photoplagios, on the basis of these apomorphic LOS features and also present derived features of the LOS to diagnose several additional leiognathid clades, including Gazza and Secutor. Furthermore, we show that five distinct and highly specialized morphologies for male-specific lateral luminescence signaling, which exhibit species-specific variation in structure, have evolved in these otherwise outwardly conservative fishes. Leiognathids inhabit turbid coastal waters with poor visibility and are often captured in mixed assemblages of several species. We hypothesize that the species-specific, sexually dimorphic internal and external modifications of the leiognathid LOS provide compelling evidence for an assortative mating scheme in which males use species-specific patterns of lateral luminescence signaling to attract mates, and that this system functions to maintain reproductive isolation in these turbid coastal environments.

Patterning across the ascidian neural plate by lateral Nodal signalling sources

Ascidians are invertebrate chordates with a simple larval tadpole form containing a notochord and an overlying dorsal neural tube. As in vertebrates, the neural tube of ascidian larvae displays positional differences along the rostral-caudal and dorsal-ventral axes in terms of neuronal cell types generated, morphology and gene expression. However, how these differences are established in this simple chordate remains largely unknown. In this study, we show that a single blastomere named b6.5, which is situated in a lateral position in the 32-cell-stage embryo, is a source of signal(s) required for patterning across the medial-lateral axis (future ventral-dorsal axis) of the neural plate. We identify this signal as a Ciona homologue of Nodal, Ci-Nodal. Transcriptional activation of Ci-Nodal in b6.5 depends upon vegetally derived Ci-FGF9/16/20. Using three distinct reagents to inhibit Nodal signals, we show that Nodal signalling is required for neural plate patterning across the medial-lateral axis and that, in the absence of this signal, the caudal-lateral part of the neural plate adopts a medial-like fate. Secondary muscle fate is similarly affected. We conclude that specification of the lateral neural plate is initiated by signalling sources laterally flanking the neural plate and involves a cell-fate choice between lateral and medial neural fates, with Nodal signalling promoting lateral fate. This role for Nodal signalling during ascidian neural plate patterning contrasts with that in vertebrates, where it is implicated in promoting a medial neural fate, the floor plate.

Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit

Microcircuits composed of principal neuron and interneuron dendrites have an important role in shaping the representation of sensory information in the olfactory bulb. Here we establish the physiological features governing synaptic signaling in dendrodendritic microcircuits of olfactory bulb glomeruli. We show that dendritic gamma-aminobutyric acid (GABA) release from periglomerular neurons mediates inhibition of principal tufted cells, retrograde inhibition of sensory input and lateral signaling onto neighboring periglomerular cells. We find that L-type dendritic Ca(2+) spikes in periglomerular cells underlie dendrodendritic transmission by depolarizing periglomerular dendrites and activating P/Q type channels that trigger GABA release. Ca(2+) spikes in periglomerular cells are evoked by powerful excitatory inputs from a single principal cell, and glutamate release from the dendrites of single principal neurons activates a large ensemble of periglomerular cells.

Vulval development

C. elegans vulval development is an intensively studied example of animal organogenesis. A network of intercellular signaling, signal transduction, and transcriptional regulation underlies the precise formation of this organ, which is the connection between the hermaphrodite uterus and the outside of the nematode. A single cell of the somatic gonad, the anchor cell, organizes the development of the vulva from epidermal precursors as well as the physical connection of the epidermis with the uterus. WNT signaling acting via the HOX gene lin-39 renders six epidermal precursor cells competent to respond to other developmental signals. The anchor cell induces nearby epidermal precursor cells to generate vulval cells via an epidermal growth factor (EGF) signaling pathway. The precise pattern of vulval precursor cell fates involves the graded action of the EGF signaling and LIN-12 (Notch) mediated lateral signaling. EGF promotes the primary fate while LIN-12 promotes the secondary fate. Both EGF and LIN-12 prevent precursor cells from adopting the tertiary fate, which generates non-specialized epidermis. EGF-receptor and Notch signaling are antagonistic: EGF-receptor signaling leads to down-regulation of the Notch-like receptor LIN-12, while LIN-12 signaling induces negative regulators of EGF-receptor signaling such as MAP kinase phosphatase LIP-1 and the tyrosine kinase ARK-1. The primary precursor cell generates vulE and vulF mature vulval cells; the pattern of vulE and vulF cells requires an additional signal from the anchor cell as well as WNT signaling. The two secondary precursor cells generate vulA, vulB1, vulB2, vulC and vulD cells but in mirror symmetric polar patterns: ABCD and DCBA. The reversed polarity of the posterior secondary precursor cell lineage requires WNT signaling mediated by both Frizzled class and Ryk class WNT-receptors LIN-17 and LIN-18, respectively. A network of transcription factors controls the seven mature adult cell types; these include the LIM domain protein LIN-11, the Pax2/5/8 protein EGL-38, the zinc finger protein LIN-29, and the Nkx6.1/6.2 protein COG-1. The anchor cell also patterns nearby uterine cells, via the DSL ligand LAG-2 and LIN-12, to generate the four uv1 cells that form the tight connection with the vulva. This connection is initiated by the anchor cell, which invades between the vulF cells in a process analogous to invasive behavior of metastatic tumor cells. During this invasion process, the basement membranes between the gonad and body wall are degraded. The extensive information about vulval development in C. elegans has helped it become a paradigmatic case for identifying and studying a variety of regulatory pathways.

A Toll-interleukin 1 repeat protein at the synapse specifies asymmetric odorant receptor expression via ASK1 MAPKKK signaling

A stochastic lateral signaling interaction between two developing Caenorhabditis elegans AWC olfactory neurons causes them to take on asymmetric patterns of odorant receptor expression, called AWC(OFF) and AWC(ON). Here we show that the AWC lateral signaling gene tir-1 (previously known as nsy-2) encodes a conserved post-synaptic protein that specifies the choice between AWC(OFF) and AWC(ON). Genetic evidence suggests that tir-1 acts downstream of a voltage-gated calcium channel and CaMKII (UNC-43) to regulate AWC asymmetry via the NSY-1(ASK1) p38/JNK MAP (mitogen-activated protein) kinase cascade. TIR-1 localizes NSY-1 to post-synaptic regions of AWC, and TIR-1 binds UNC-43, suggesting that it assembles a synaptic signaling complex that regulates odorant receptor expression. Temperature-shift experiments indicate that tir-1 affects AWC during a critical period late in embryogenesis, near the time of AWC synapse formation. TIR-1 is a multidomain protein with a TIR (Toll-interleukin-1 receptor) domain that activates signaling, SAM repeats that mediate localization to post-synaptic regions of axons, and an N-terminal inhibitory domain. TIR-1 and other TIR proteins are implicated in vertebrate and invertebrate innate immunity, as are NSY-1/ASK1 kinases, so this pathway may also have a conserved function in immune signaling.

Lateral Hypothalamic Signaling Mechanisms Underlying Feeding Stimulation: Differential Contributions of Src Family Tyrosine Kinases to Feeding Triggered Either by NMDA Injection or by Food Deprivation

In rats, feeding can be triggered experimentally using many approaches. Included among these are (1) food deprivation and (2) acute microinjection of the neurotransmitter l-glutamate (Glu) or its receptor agonist NMDA into the lateral hypothalamic area (LHA). Under both paradigms, the NMDA receptor (NMDA-R) within the LHA appears critically involved in transferring signals encoded by Glu to stimulate feeding. However, the intracellular mechanisms underlying this signal transfer are unknown. Because protein-tyrosine kinases (PTKs) participate in NMDA-R signaling mechanisms, we determined PTK involvement in LHA mechanisms underlying both types of feeding stimulation through food intake and biochemical measurements. LHA injections of PTK inhibitors significantly suppressed feeding elicited by LHA NMDA injection (up to 69%) but only mildly suppressed deprivation feeding (24%), suggesting that PTKs may be less critical for signals underlying this feeding behavior. Conversely, food deprivation but not NMDA injection produced marked increases in apparent activity for Src PTKs and in the expression of Pyk2, an Src-activating PTK. When considered together, the behavioral and biochemical results demonstrate that, although it is easier to suppress NMDA-elicited feeding by PTK inhibitors, food deprivation readily drives PTK activity in vivo. The latter result may reflect greater PTK recruitment by neurotransmitter receptors, distinct from the NMDA-R, that are activated during deprivation-elicited but not NMDA-elicited feeding. These results also demonstrate how the use of only one feeding stimulation paradigm may fail to reveal the true contributions of signaling molecules to pathways underlying feeding behavior in vivo.

Lateral diffusion of CO2 in leaves of the crassulacean acid metabolism plant Kalancho� daigremontiana Hamet et Perrier

Dynamic patchiness of photosystem II (PSII) activity in leaves of the crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana Hamet et Perrier, which was independent of stomatal control and was observed during both the day/night cycle and circadian endogenous oscillations of CAM, was previously explained by lateral CO2 diffusion and CO2 signalling in the leaves [Rascher et al. (2001) Proc Natl Acad Sci USA 98:11801-11805; Rascher and Luttge (2002) Plant Biol 4:671-681]. The aim here was to actually demonstrate the importance of lateral CO2 diffusion and its effects on localized PSII activity. Covering small sections of entire leaves with silicone grease was used for local exclusion of a contribution of atmospheric CO2 to internal CO2 via transport through stomata. A setup for combined measurement of gas exchange and chlorophyll fluorescence imaging was used for recording photosynthetic activity with a spatiotemporal resolution. When remobilization of malic acid from vacuolar storage and its decarboxylation in the CAM cycle caused increasing internal CO2 concentrations sustaining high PSII activity behind closed stomata, PSII activity was also increased in adjacent leaf sections where vacuolar malic acid accumulation was minimal as a result of preventing external CO2 supply due to leaf-surface greasing, and where therefore CO2 could only be supplied by diffusion from the neighbouring malic acid-remobilizing leaf tissue. This demonstrates lateral CO2 diffusion and its effect on local photosynthetic activity.

Cell contact-dependent mechanisms specify taste bud pattern during a critical period early in embryonic development.

After gastrulation, the pharyngeal endoderm is specified to give rise to taste receptor organs without further signaling from other embryonic tissues. We hypothesized that intercellular signaling might be responsible for the specification of taste buds. To test if and when this signaling was occurring, intercellular contacts were transiently disrupted in cultures of pharyngeal endoderm from axolotl embryos, and the number, size, and distribution of taste buds analyzed. Disruption of cell contacts at progressive time points, from neurula to late tail bud stages, revealed a critical period, during mid-tail bud stages, when disruption of cell contacts resulted in a significant increase in taste bud number and size. The spatial distribution of taste buds was also altered; taste buds were more clustered in explants disrupted during the critical period. These effects were not due to general alterations in mitosis and apoptosis. Rather, at least three aspects of taste bud patterning, i.e., number, size, and distribution, are governed by mechanisms dependent on normal cell contacts during a concise time window. Furthermore, our findings are consistent with specification of taste buds by means of lateral inhibitory signaling, which we hypothesize results from cell contact-dependent or short-range diffusible signals.

Lateral signaling enhances TGF-β response complexity

TGF-β elicits context-dependent and cell-specific effects that often appear conflicting, such as stimulation or inhibition of growth, apoptosis or differentiation. It is puzzling how such a diverse array of responses can result from binding of TGF-β to a single receptor complex that activates a seemingly straightforward signal-transduction scheme dependent on shuttling of Smad transducer proteins from the receptor to the nucleus. Here, we discuss a novel paradigm for TGF-β signaling in endothelial cells in which the same ligand can induce opposing effects mediated by activation of two different classes of Smads through a chimeric receptor complex.

The Lateral Signal for LIN-12/Notch in C. elegans Vulval Development Comprises Redundant Secreted and Transmembrane DSL Proteins

The vulval precursor cells (VPCs) are spatially patterned by a LET-23/EGF receptor-mediated inductive signal and a LIN-12/Notch-mediated lateral signal. The lateral signal has eluded identification, so the mechanism by which lateral signaling is activated has not been known. Here, we computationally identify ten genes that encode potential ligands for LIN-12, and show that three of these genes, apx-1, dsl-1, and lag-2, are functionally redundant components of the lateral signal. We also show that transcription of all three genes is initiated or upregulated in VPCs in response to inductive signaling, suggesting that direct transcriptional control of the lateral signal by the inductive signal is part of the mechanism by which these cell signaling events are coordinated. In addition, we show that DSL-1, which lacks a predicted transmembrane domain, is a natural secreted ligand and can substitute for the transmembrane ligand LAG-2 in different functional assays.

Crosstalk between the EGFR and LIN-12/Notch pathways in C. elegans vulval development.

The Caenorhabditis elegans vulva is an important paradigm for cell-cell interactions in animal development. The fates of six vulval precursor cells are patterned through the action of the epidermal growth factor receptor-mitogen-activated protein kinase (EGFR-MAPK) inductive signaling pathway, which specifies the 1 degrees fate, and the LIN-12/Notch lateral signaling pathway, which specifies the 2 degrees fate. Here, we provide evidence that the inductive signal is spatially graded and initially activates the EGFR-MAPK pathway in the prospective 2 degrees cells. Subsequently, this effect is counteracted by the expression of multiple new negative regulators of the EGFR-MAPK pathway, under direct transcriptional control of the LIN-12-mediated lateral signal.

The two steps of vulval induction in Oscheius tipulae CEW1 recruit common regulators including a MEK kinase

The cell interactions that specify the spatial pattern of vulval precursor cell (VPC) fates differ between the nematodes Oscheius tipulae CEW1 and Caenorhabditis elegans. In the former, the centered pattern of fates is obtained by two successive inductions from the gonadal anchor cell, whereas in the latter, a single inductive step by the anchor cell (EGF–Ras–MAP kinase pathway) can act as a morphogen and is reinforced by lateral signaling between the vulval precursors (Notch pathway). We performed a genetic screen for vulva mutants in O. tipulae CEW1. Here we present the mutants that specifically affect the vulval induction mechanisms. Phenotypic and epistatic analyses of these mutants show that both vulval induction steps share common components, one of which appears to be MEK kinase(s). Moreover, the inductive pathway (including MEK kinase) influences the competence of the vulval precursor cells and more strikingly their division pattern as well, irrespective of their vulval fate. Finally, a comparison of vulval mutant phenotypes obtained in C. elegans and O. tipulae CEW1 highlights the evolution of vulval induction mechanisms between the two species.

Thigh pain due to intraspinal neurilemmoma

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How do cells know what they want to be when they grow up? Lessons from epidermal patterning in Arabidopsis.

Because the plant epidermis is readily accessible and consists of few cell types on most organs, the epidermis has become a well-studied model for cell differentiation and cell patterning in plants. Recent advances in our understanding of the development of three epidermal cell types, trichomes, root hairs, and stomata, allow a comparison of the underlying patterning mechanisms. In Arabidopsis, trichome development and root epidermal patterning use a common mechanism involving closely related cell fate transcription factors and a similar lateral inhibition signaling pathway. Yet the resulting patterns differ substantially, primarily due to the influence of a prepattern derived from subepidermal cortical cells in root epidermal patterning. Stomatal patterning uses a contrasting mechanism based primarily on control of the orientation of cell divisions that also involves an inhibitory signaling pathway. This review focuses on comparing and contrasting these patterning pathways to identify and illustrate general themes that may be broadly applicable to other systems. Where these pathways occur in the same tissue, interaction and competition between these pathways is also discussed.

Microvillar Membrane Microdomains Exist at Physiological Temperature: ROLE OF GALECTIN-4 AS LIPID RAFT STABILIZER REVEALED BY “SUPERRAFTS”

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.

Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans.

The coordination of signals from different pathways is important for cell fate specification during animal development. Here, we define a novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p (ref. 2), the VPC believed to be the source of the lateral signal. Here we identify a 'downregulation targeting signal' (DTS) in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. We also show that if LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo.

Accolade for elegans

Accolade for elegans