Outermost Cell Layer Research Articles

ATML1 promotes epidermal cell differentiation in Arabidopsis shoots

Molecular mechanisms that generate distinct tissue layers in plant shoots are not well understood. ATML1, an Arabidopsis homeobox gene, is expressed in the outermost cell layer, beginning at an early stage of development. The promoters of many epidermis-specific genes, including ATML1, contain an ATML1-binding site called an L1 box, suggesting that ATML1 regulates epidermal cell fate. Here, we show that overexpression of ATML1 was sufficient to activate the expression of epidermal genes and to induce epidermis-related traits such as the formation of stomatal guard cells and trichome-like cells in non-epidermal seedling tissues. Detailed observation of the division planes of these ectopic stomatal cells suggested that a near-surface position, as well as epidermal cell identity, were required for regular anticlinal cell division, as seen in wild-type epidermis. Moreover, analyses of a loss-of-function mutant and overexpressors implied that differentiation of epidermal cells was associated with repression of mesophyll cell fate. Collectively, our studies contribute new information about the molecular basis of cell fate determination in different layers of plant aerial organs.

A New Model of Development of the Mammalian Ovary and Follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.

Defects in leaf epidermis of Arabidopsis thaliana plants with CDKA;1 activity reduced in the shoot apical meristem

In Arabidopsis thaliana, like in other dicots, the shoot epidermis originates from protodermis, the outermost cell layer of shoot apical meristem. We examined leaf epidermis in transgenic A. thaliana plants in which CDKA;1.N146, a negative dominant allele of A-type cyclin-dependent kinase, was expressed from the SHOOTMERISTEMLESS promoter, i.e., in the shoot apical meristem. Using cleared whole mount preparations of expanding leaves and sequential in vivo replicas of expanding leaf surface, we show that dominant-negative CDKA;1 expression results in defects in epidermis continuity: loss of individual cells and occurrence of gaps between anticlinal walls of neighboring pavement cells. Another striking feature is ingrowth-like invaginations of anticlinal cell walls of pavement cells. Their formation is related to various processes: expansion of cells surrounding the sites of cell loss, defected cytokinesis, and presumably also, the actual ingrowth of an anticlinal cell wall. The mutant exhibits also increased variation in cell size and locally reduced waviness of anticlinal walls of pavement cells. These unusual features of leaf epidermis phenotype may shed a new light on our knowledge on morphogenesis of jigsaw puzzle-shaped pavement cells and on the CDKA;1 role in regulation of plant development via influence on cytoskeleton and plant cell wall.

ONION2 Fatty Acid Elongase is Required for Shoot Development in Rice

A plant’s surface is covered with epicuticular wax, which protects plants from inappropriate environmental conditions such as drought and pathogen attack. Very-long-chain fatty acids (VLCFAs) are the main component of epicuticular wax on the surface of above-ground organs. Here we show that a fatty acid elongase catalyzing an elongation reaction of VLCFAs is required for shoot development in rice. onion2 (oni2) mutants produced very small shoots in which leaves were fused to each other, and ceased growing after germination. The midrib of oni2 leaf blades was not developed correctly. Molecular cloning showed that ONI2 encodes a fatty acid elongase, which catalyzes the first step of elongation reactions of a carbon chain of VLCFAs, and oni2 had a reduced amount of VLCFAs. Expression analysis showed that ONI2 is specifically expressed in the outermost cell layer of young lateral organs. These results suggest that ONI2 is a layer 1-specific gene required for development of the entire shoot and that VLCFAs play an essential role in normal shoot development in rice.

Engineering Islets for Improved Performance by Optimized Reaggregation in a Micromold

Isolated islets can provide a source of tissue for research, transplantation, and drug discovery to develop therapies for diabetes. Empirical modeling of islet diffusion barriers demonstrated that only the outermost layers of cells were exposed to glucose and sufficient oxygen levels, resulting in core cell death. Islets under a diameter of 100 μm exhibited a lower diffusion barrier, superior survival rates, and improved functional properties. Utilizing these observations, we engineered optimal islets by dispersing them into single cells and reaggregating them over several days in a micromold. These custom-designed micromolds contained conical-shaped recesses that enhanced reaggregation of cells into a defined geometry. The engineered islets, or Kanslets, were all under 100 μm in diameter, and had the same general cellular composition as native islets. Kanslets continued to produce new insulin molecules and had microvilli on the islet surface, much like native islets. The engineered islets had a statistically higher viability (percent of live cells), and increased glucose diffusion compared to native islets. In addition, they remained responsive to varying glucose levels by secreting insulin. When transplanted into diabetic rats, engineered islets performed reduced random blood glucose to normal levels within 48 h. Optimally, engineering islets may be a suitable alternative to utilizing native, isolated islet tissue for a variety of applications. Reaggregating tissue in an optimized manner using our engineered micromold approach has immense impact for three-dimensional tissue production and its subsequent use in research, drug discovery, and the clinic.

Differentiation of endosperm transfer cells of barley: a comprehensive analysis at the micro‐scale

Barley endosperm cells differentiate into transfer cells (ETCs) opposite the nucellar projection. To comprehensively analyse ETC differentiation, laser microdissection-based transcript and metabolite profiles were obtained from laser microdissected tissues and cell morphology was analysed. Flange-like secondary-wall ingrowths appeared between 5 and 7 days after pollination within the three outermost cell layers. Gene expression analysis indicated that ethylene-signalling pathways initiate ETC morphology. This is accompanied by gene activity related to cell shape control and vesicle transport, with abundant mitochondria and endomembrane structures. Gene expression analyses indicate predominant formation of hemicelluloses, glucuronoxylans and arabinoxylans, and transient formation of callose, together with proline and 4-hydroxyproline biosynthesis. Activation of the methylation cycle is probably required for biosynthesis of phospholipids, pectins and ethylene. Membrane microdomains involving sterols/sphingolipids and remorins are potentially involved in ETC development. The transcriptional activity of assimilate and micronutrient transporters suggests ETCs as the main uptake organs of solutes into the endosperm. Accordingly, the endosperm grows maximally after ETCs are fully developed. Up-regulated gene expression related to amino acid catabolism, C:N balances, carbohydrate oxidation, mitochondrial activity and starch degradation meets high demands for respiratory energy and carbohydrates, required for cell proliferation and wall synthesis. At 10 days after pollination, ETCs undergo further differentiation, potentially initiated by abscisic acid, and metabolism is reprogrammed as shown by activated storage and stress-related processes. Overall, the data provide a comprehensive view of barley ETC differentiation and development, and identify candidate genes and associated pathways.

English

Chrysomya megacephala (F.) (Diptera: Calliphoridae) is a medically important blow fly species in many parts of the world. In this study, we examined the ovary of C. megacephala at the ultrastructural level using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Dissection of the ovaries revealed eight stages of ovarian development, thus providing a means of estimation of the age of females based on this organ. In the initial stage I (day 0-2), freshly-emerged flies up to three days old each display piriform germaria while the follicles are not yet well differentiated. In stage II (day 3), each follicle is nearly spherical and sepa- rating from the germarium. During stage III (day 4), each follicle is now easily distinguished from the germarium and is only connected by a thin interfollicular stalk. The nurse cells are completely surrounded by the cuboidal epithelial follicular cells. In stage IV (day 5-6), each folli- cle is spherical and considerably enlarged; whereas, the nurse cells within are more differentiated into individual round inclusions and grouped in the follicle chamber. Stage V (day 7) marks appearance of the visible oocyte at the posterior pole of the follicle. During stage VI (day 8), each follicle displays oocyte expansion anteriorly to occupyfrom one-third to one-half of the total length. In stage VII (day 9) the oo- cyte characteristically fills the posterior two-thirds of the total length of the follicle and in the final stage VIII (day 10), each follicle is com- pletely filled by an oocyte and nurse cells have disappeared. The micropyle and narrow, elongate median area are clearly seen in this final stage. SEM images reveal that the ovary is covered by an ovarian envelope, which is tough and thickened in 3-day-old adult ovarioles; whereas, it becomes thin and fragile in 9-day-old adult ovarioles. TEM analysis of ovarioles of 3-day-old adult flies shows that cuboidal follic- ular cells containing large nuclei with visible fibrils of muscle simply encircle the large compartments of nurse cells; whereas, ovarioles of 7- day-old adult flies displayed a more completely developed enclosure for the developing oocyte. This was observed by the presence of the outermost follicular cell layer with underlying eggshell, vitelline envelope, trabecular layer, and layer of granular material that surrounded the innermost oocyte.

Relationship between Pericarp Structure and Kernel Tenderness in Super Sweet Corn

In order to understand the relationship between kernel tenderness and pericarp structure, histological studies of a set of super-sweet corn (Z. mays subsp. mays) inbred lines (T4, T5, T19, T38, and T39) kernels at different developmental stages were conducted. The result indicated that super-sweet corn pericarp was formed with several layers of cells, where the outermost layer of cells packed tightly with horny cell walls. T38 and T39 inbred lines had a good eating qualities due to their thinner cell walls and less cell layers of pericarp combining with lower level of fibrosis and lignification of cell wall. On the contrary, T4 and T5 inbred lines did not have a eating qualities with thicker cell walls and more cell layers of pericarp combining with high level of fibrosis and lignification of cell wall. T19 had the worst taste for its high-density pericarp cells adding very high fibrosis and lignification of cell wall among the five inbred lines.

Raphides in the Uncalcified Siphonous Green Seaweed,Codium minus(Schmidt) P. C. Silva

The vacuole of utricles, the outermost cell layer of the siphonous green seaweed,Codium minus, had numerous single needles and needle bundles. The crystals composing each needle appeared arranged in a twisted configuration, both ends were pointed, and each needle was contained in a matrix or membrane; bundles of needles appeared enclosed by a matrix. Chemical and electron diffraction analysis indicated that the needles consisted of calcium oxalate. This is the first paper on terrestrial plant-like raphides in an alga.

A sequential study of incomplete Freund’s adjuvant-induced peritonitis in Atlantic cod

Development of diagnostic and prophylactic methodologies is dependent on knowledge of the host’s defence system and reaction to different vaccine adjuvants. Here we present a sequential morphological study of peritonitis and inflammatory cell processing of incomplete Freund’s adjuvant (IFA) in intraperitoneally injected Atlantic cod. The peritoneal tissue responses were characterised using necropsy, histology and electron microscopy. An extensive inflammatory response as characterised by leukocyte morphology and contents of enzymes, presence of apoptotic cells and IFN-γ-expressing cells was observed. Three days post injection, IFA droplets were surrounded by different types of inflammatory cells and two different patterns could be discerned. The first was characterised by flattened and concentrically arranged interdigitating cells connected by desmosomes and with macrophage-like cells (MLCs) predominant in the periphery. The second type possessed four stratified layers with an inner layer containing many apoptotic MLCs; a second layer containing flattened and shrunken cells and outer layers comprising moderately flattened cells and an outermost layer of mononuclear cells expressing IFN-γ. Oil was detected both inside and outside MLCs. The two types of processes, of which the second was clearly stratified, were similar to those observed in other teleosts, indicating a variety of reaction modes or alternatively sequential process development. The numerous dead MLCs contributed to inflammation.

Male cone morphogenesis, pollen development and pollen dispersal mechanism in Ginkgo biloba L.

Lu, Y., Wang, L., Wang, D., Wang, Y., Zhang, M., Jin, B. and Chen, P. 2011. Male cone morphogenesis, pollen development and pollen dispersal mechanism inGinkgo bilobaL. Can. J. Plant Sci. 91: 971-981. Ginkgo biloba L. is one of the oldest gymnosperms. Male cone morphogenesis, pollen development and dispersal are important for successful pollination and reproduction. In this study, we investigated the development of male cone, pollen and the sporangial wall in detail. The results indicate that: (1) The primordia of male cones and leaves begin to differentiate in early June and remain open until the following March. The male cones then mature and release pollen in mid-April. The male cones are drooped and approximately perpendicular to the leaves during pollination. (2) The microsporocytes develop from the sporogenous cell and form a tetrahedral tetrad after two simultaneous asymmetrically meioses, then produce a matured four-cell pollen after three polar mitotic divisions. The matured pollen is hemispheric in shape with a large aperture area and three pollen wall layers; once released from the microsporangia, the pollen becomes boat-like in shape. (3) The sporangial walls are eusporangiate and consist of epidermis, endothecium and tapetum. The differentiation of the tapetum occurs separately from that of the epidermis and endothecium, and originates from the outermost layer of sporogenous cells. The sporangial walls exhibit shrinkage of the epidermis, fibrous thickening of the endothecium, and enzymic dissolution of the tapetum during pollen dispersal, which contributes to microsporangia opening. Based on these results, we conclude that there many unique and primitive characteristics of the development of the male cones, pollen and sporangial wall of G. biloba. In addition, we also found that the male cones, pollen and sporangial walls have evolved efficient structural and morphological adaptations to anemophily.

Male cone morphogenesis, pollen development and pollen dispersal mechanism in Ginkgo biloba L.

Lu, Y., Wang, L., Wang, D., Wang, Y., Zhang, M., Jin, B. and Chen, P. 2011. Male cone morphogenesis, pollen development and pollen dispersal mechanism in Ginkgo biloba L. Can. J. Plant Sci. 91: 971–981. Ginkgo biloba L. is one of the oldest gymnosperms. Male cone morphogenesis, pollen development and dispersal are important for successful pollination and reproduction. In this study, we investigated the development of male cone, pollen and the sporangial wall in detail. The results indicate that: (1) The primordia of male cones and leaves begin to differentiate in early June and remain open until the following March. The male cones then mature and release pollen in mid-April. The male cones are drooped and approximately perpendicular to the leaves during pollination. (2) The microsporocytes develop from the sporogenous cell and form a tetrahedral tetrad after two simultaneous asymmetrically meioses, then produce a matured four-cell pollen after three polar mitotic divisions. The matured pollen is hemispheric in shape with a large aperture area and three pollen wall layers; once released from the microsporangia, the pollen becomes boat-like in shape. (3) The sporangial walls are eusporangiate and consist of epidermis, endothecium and tapetum. The differentiation of the tapetum occurs separately from that of the epidermis and endothecium, and originates from the outermost layer of sporogenous cells. The sporangial walls exhibit shrinkage of the epidermis, fibrous thickening of the endothecium, and enzymic dissolution of the tapetum during pollen dispersal, which contributes to microsporangia opening. Based on these results, we conclude that there many unique and primitive characteristics of the development of the male cones, pollen and sporangial wall of G. biloba. In addition, we also found that the male cones, pollen and sporangial walls have evolved efficient structural and morphological adaptations to anemophily.

Analysis of Gene Expression Patterns during Seed Coat Development in Arabidopsis

The seed coat is important for embryo protection, seed hydration, and dispersal. Seed coat composition is also of interest to the agricultural sector, since it impacts the nutritional value for humans and livestock alike. Although some seed coat genes have been identified, the developmental pathways controlling seed coat development are not completely elucidated, and a global genetic program associated with seed coat development has not been reported. This study uses a combination of genetic and genomic approaches in Arabidopsis thaliana to begin to address these knowledge gaps. Seed coat development is a complex process whereby the integuments of the ovule differentiate into specialized cell types. In Arabidopsis, the outermost layer of cells secretes mucilage into the apoplast and develops a secondary cell wall known as a columella. The layer beneath the epidermis, the palisade, synthesizes a secondary cell wall on its inner tangential side. The innermost layer (the pigmented layer or endothelium) produces proanthocyanidins that condense into tannins and oxidize, giving a brown color to mature seeds. Genetic separation of these cell layers was achieved using the ap2-7 and tt16-1 mutants, where the epidermis/palisade and the endothelium do not develop respectively. This genetic ablation was exploited to examine the developmental programs of these cell types by isolating and collecting seed coats at key transitions during development and performing global gene expression analysis. The data indicate that the developmental programs of the epidermis and the pigmented layer proceed relatively independently. Global expression datasets that can be used for identification of new gene candidates for seed coat development were generated. These dataset provide a comprehensive expression profile for developing seed coats in Arabidopsis, and should provide a useful resource and reference for other seed systems.

Altered expression of auxin-related genes in the fatty acid elongase mutant oni1 of rice

VLCFAs are the main components of cuticular wax, which covers and protects plants from physical and biological stresses. However, the effect of fatty acid composition or the physiological role of VLCFAs on plant development under normal growth conditions is not well understood. We analyzed loss-of-function mutants of ONION1 (ONI1) which encodes fatty acid elongase (β-ketoacyl CoA synthase) catalyzing an elongation reaction of a carbon chain of VLCFAs. We showed that oni1 shoot contained a reduced amount of VLCFAs, and differentiation and functionality of an outermost cell layer (L1) were highly perturbed in oni1 shoot. In spite of the L1-specific expression of ONI1, the effects of the oni1 mutation were not restricted to L1, but expanded to inner cells, so that the entire shoot development was impaired including failure of the maintenance of the SAM and ectopic expression of SAM-specific KNOX genes in leaf. Thus, ONI1 function is cell non-autonomous, and signaling from L1 to inner cells may support proper development of inner cells. Here we report that expression of auxin-related genes was affected in oni1 shoot, and we speculate the existence of improper auxin distribution due to a lack of normal L1 in oni1 shoot.

Chitosan-reduced gold nanoparticles: a novel carrier for the preparation of spray-dried liposomes for topical delivery

Exposure of skin to various chemical and physical agents results in excessive stress to the outermost cell layer of the skin, causing different degenerative effects that can be minimized by using antioxidant formulations. The major challenge, in this regard, is to develop a formulation, which can prevent photodegradation of the actives, thus allowing a significant amount to be deposited at the site. In recent decades, liposomal formulations have been extensively employed to overcome the barrier properties of the skin and photodegradation of actives. In the present study, chitosan-reduced gold nanoparticles were investigated for its potential as a carrier to prepare liposomes by a spray-drying method. Liposomes so obtained were characterized for phospholipid recovery, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, particle size, zeta potential, encapsulation efficiency, and deposition of drug and gold nanoparticles in the rat skin. Further, a liposomal gel formulation was prepared using Carbopol® 980 NF (Noveon Systems, Kochi, India) and evaluated for drug deposition in the skin. Antioxidant activity of vitamin C encapsulated in gold liposomes was determined on a human leukemia (HL-60) cell line. The use of gold nanoparticles as a carrier showed improved phospholipid recovery and thus overcomes the liposome scalability problem. DRIFT spectra confirmed the presence of phospholipid in the formulation. Liposomal gel showed improved drug deposition, as compared to control and marketed preparations. A more interesting contribution of the chitosan-reduced gold nanoparticles was an enhanced antioxidant activity seen in case of the vitamin C–loaded gold liposomal formulation. Liposomal formulation was found to be stable for 3 months at 30°C and 65% relative humidity.

Fatty acid elongase is required for shoot development in rice

Organisms are covered extracellularly with cuticular waxes that consist of various fatty acids. In higher plants, extracellular waxes act as indispensable barriers to protect the plants from physical and biological stresses such as drought and pathogen attacks. However, the effect of fatty acid composition on plant development under normal growth conditions is not well understood. Here we show that the ONION1 (ONI1) gene, which encodes a fatty acid elongase (β-ketoacyl CoA synthase) involved in the synthesis of very-long-chain fatty acids, is required for correct fatty acid composition and normal shoot development in rice. oni1 mutants containing a reduced amount of very-long-chain fatty acids produced very small shoots, with an aberrant outermost epidermal cell layer, and ceased to grow soon after germination. These mutants also showed abnormal expression of a KNOX family homeobox gene. ONI1 was specifically expressed in the outermost cell layer of the shoot apical meristem and developing lateral organs. These results show that fatty acid elongase is required for formation of the outermost cell layer, and this layer is indispensable for entire shoot development in rice.

Monte Carlo Simulation of Electron Dose from 131I-Targeted Tumor Cells Within a Heterogeneous Tumor

In internal radiotherapy, the variable distribution of target receptors within the tumoral tissue, and the variable ranges of electrons may be responsible for a heterogeneous dose distribution at the cellular level. The aim of the present study was to use Monte Carlo simulations to assess (131)I electron dose in a model of heterogeneous tumor containing multiple clusters of cancer cells, targeted by (131)I-labeled molecules. The model consisted of 150-μm-diameter spherical tumor cell clusters, in which (131)I was homogeneously distributed. Clusters were placed 24 μm apart, separated by septa of nonradioactive connective tissue. The electron dose distribution to tumor cells in a single cluster was first assessed. Then was assessed the dose increase to these targets after adding multiple layers of neighboring clusters (total number of clusters = 15,624). Dose distribution within a single isolated cluster follows a decreasing gradient, the dose for the outermost cell layer being about half that at the center. When radioactive neighbors were added, the dose to the central cluster increased. The most important contribution was given by the nearest neighbors, whereas the contribution from neighbors beyond a distance of 1 mm was only for 5% of the final dose. If the central cluster was unlabeled, the absorbed dose to the outermost cell layer of this cluster was reduced by 27%, and that at the center by 45%. The electron cross-dose of (131)I falls rapidly as a function of distance and becomes negligible after just 1 mm. Small clusters of tumor cells that are not radiolabeled may receive a very small dose. Therefore, in internal radiotherapy it is important to aim at targeting tumor cells as homogeneously as possible, rather than relying on the cross-dose to achieve a therapeutic effect.

The allergy gene: how a mutation in a skin protein revealed a link between eczema and asthma

Ichthyosis vulgaris is a common genetic skin disorder characterized by dry, scaly skin. About 1% of the European population have the full presentation of ichthyosis vulgaris; up to 10% have a milder, subclinical form. Atopic eczema is the most common, inflammatory skin condition, affecting 20% of children. It is often accompanied by a number of other allergies, including atopic asthma. Atopic eczema is a complex trait, where predisposing genes in combination with environmental stimuli produce the disease. Recently, we reported the first loss-of-function genetic mutations in the filaggrin gene as the cause of ichthyosis vulgaris. We noted people with ichthyosis vulgaris also have atopic eczema (and vice versa) and that the filaggrin gene sits in a known atopic eczema susceptibility locus. We went on to confirm that filaggrin mutations, carried by up to 10% of the population, are the major genetic predisposing factor for atopic eczema and the various allergies associated with atopic eczema. Filaggrin is a highly abundant protein expressed in the uppermost part of the epidermis that is critical to the formation and hydration of the stratum corneum—the outermost dead cell layers responsible for the barrier function of the skin. Filaggrin deficiency leads to a “leaky” skin barrier that allows higher than normal water loss (explaining the dry, scaly skin), as well as allowing entry of allergens through the epidermis where they trigger inflammatory and allergic immune responses (atopic eczema and allergies). This work has placed the skin barrier at the center stage of eczema and allergy research and has kick-started new therapy development programs aimed at repairing or enhancing skin-barrier function as a means of treating or preventing these very common diseases.

Chemosensory function of amphibian skin: integrating epithelial transport, capillary blood flow and behaviour

Terrestrial anuran amphibians absorb water across specialized regions of skin on the posterioventral region of their bodies. Rapid water absorption is mediated by the insertion of aquaporins into the apical membrane of the outermost cell layer. Water moves out of the epithelium via aquaglyceroporins in the basolateral membrane and into the circulation in conjunction with increased capillary blood flow to the skin and aquaporins in the capillary endothelial cells. These physiological responses are activated by intrinsic stimuli relating to the animals' hydration status and extrinsic stimuli relating to the detection of osmotically available water. The integration of these processes has been studied using behavioural observations in conjunction with neurophysiological recordings and studies of epithelial transport. These studies have identified plasma volume and urinary bladder stores as intrinsic stimuli that activate the formation of angiotensin II (AII) to stimulate water absorption behaviour. The coordinated increase in water permeability and capillary blood flow appears to be mediated primarily by sympathetic stimulation of beta adrenergic receptors, although the neurohypopyseal hormone arginine vasotocin (AVT) may also play a role. Extrinsic stimuli relate primarily to the ionic and osmotic properties of hydration sources. Toads avoid NaCl solutions that have been shown to be harmful in acute exposure, approx. 200-250 mm. The avoidance is partially attenuated by amiloride raising the hypothesis that the mechanism for salt detection by toads resembles that for salt taste in mammals that take in water by mouth. In this model, depolarization of the basolateral membrane of taste cells is coupled to afferent neural stimulation. In toad skin we have identified innervation of skin epithelial cells by branches of spinal nerves and measured neural responses to NaCl solutions that elicit behavioural avoidance. These same concentrations produce depolarization of the basolateral membrane in isolated epithelial preparations. As with salt taste in mammals, the neural responses and depolarization of basolateral membrane potential are partially inhibited by amiloride. In addition, toads are more tolerant of sodium gluconate solution which is consistent with the phenomenon in mammalian taste physiology termed the anion paradox in which sodium salts with larger molecular weight anions produce a reduced intensity of salt taste. Finally, toads also avoid concentrated solutions of a non-electrolyte, mannitol, which differs from NaCl solutions in not affecting transepithelial conductance and requires a longer time to depolarize the basolateral membrane. Osmotic stimuli may mediate sensory processes for longer term detection of conditions with low water potential while ionic stimuli are more important for shorter term analysis of rehydration sources.

Cell Polarity: Lateral Perspectives

The outer and inner (lateral) plasma membranes of the outermost cell layer in plants provide selective barriers to the environment. Recent studies provide perspectives on how asymmetric protein localization is established at lateral membranes.