Cytological and cytochemical investigations of development from dormant gemmules of the marine sponge,Haliclona loosanoffi

Vernalized gemmules of the marine sponge Haliclona loosanoffi were cultured at 20°C, fixed at 24-hour intervals (0–11 days), and processed for light microscopy by using a variety of absorption and fluorescent staining methods. The cytochemistry and morphology of development were compared to the well-studied developmental patterns of freshwater sponges and to the patterns described in the marine sponge Suberites domuncula. The precocious development of H. loosanoffi gemmules involves early morphogenesis occurring within the unhatched gemmule, as opposed to the patterns in freshwater sponges, where most development occurs after the gemmule hatches. Definitive sponge tissue surrounding a single osculum is present 9 days after release from dormancy.     

Identification of emergent motion compartments in the amniote embryo

Abstract

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ～400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen's node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen's node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

Identification of emergent motion compartments in the amniote embryo

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ∼400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen''s node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen''s node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. I. Evidence for androgen-regulated proteolytic processing

The guinea pig seminal vesicle epithelium synthesizes and secretes four major secretory proteins (SVP-1-4). Previous work has established that these four proteins are cleaved from two primary translation products in a complex series of protein processing reactions. The present studies suggest that these protein processing reactions are regulated by androgens. In vitro labeling of seminal vesicle proteins revealed significant differences in the patterns of secretory protein intermediates produced by tissue from intact and castrated animals. Seminal vesicle tissue explants from castrated animals secreted a subset of the processing intermediates secreted by tissue from intact animals. The changes in the patterns of secretory protein intermediates became more pronounced with increasing time after castration, and were fully reversible by treatment of castrated animals with testosterone, suggesting that androgens were affecting the processing or secretion of secretory protein precursors. Amino-terminal protein sequencing of secretory protein processing intermediates that accumulate in the seminal vesicle lumen after castration suggests that the guinea pig seminal vesicle contains an androgen-regulated proteolytic processing activity.     

Effect of ovarian tissue vitrification method on mice preantral follicular development and gene expression

Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group.     

Changes in leaf organisation, photosynthetic performance and wood formation during ex vitro acclimatisation of black mulberry (Morus nigra L.)

Changes in anatomical organisation of the leaf, photosynthetic performance and wood formation were examined to evaluate the temporal and spatial patterns of acclimatisation of micropropagated slow-growing black mulberry ( Morus nigra L.) plantlets to the ex vitro environment. Leaf structure differentiation, the rates of net photosynthesis (P_n), transpiration (E) and stomatal conductance (g_s), and secondary xylem growth were determined in the course of a 56-day acclimatisation. Differentiation of palisade parenchyma was observed 7 days after transfer. At this stage, the rates of P_n, E and g_s reached maximum values, after which the rates of all three gas exchange parameters gradually decreased. The highest proportion of woody area occupied by vessels was also observed 7 days after transfer. An important feature of developing woody tissue is the difference in patterns of vessel distribution from the characteristic differentiation patterns of earlywood and latewood vessels in mature wood of ring-porous trees. Vessels with lumen areas over 3000 μm² were only differentiated in acclimatised plantlets, whereas vessels in stems sampled on days 0 and 7 had very small lumen areas of up to 560 μm². Full acclimatisation, observed 56 days after transfer to the ex vitro environment, was associated with the rapid growth of new in vivo formed leaves, very low rates of E and g_s, and much increased secondary xylem tissue within the stem area.     

Correlation-maximizing surrogate gene space for visual mining of gene expression patterns in developing barley endosperm tissue

Background  

Micro- and macroarray technologies help acquire thousands of gene expression patterns covering important biological processes during plant ontogeny. Particularly, faithful visualization methods are beneficial for revealing interesting gene expression patterns and functional relationships of coexpressed genes. Such screening helps to gain deeper insights into regulatory behavior and cellular responses, as will be discussed for expression data of developing barley endosperm tissue. For that purpose, high-throughput multidimensional scaling (HiT-MDS), a recent method for similarity-preserving data embedding, is substantially refined and used for (a) assessing the quality and reliability of centroid gene expression patterns, and for (b) derivation of functional relationships of coexpressed genes of endosperm tissue during barley grain development (0–26 days after flowering).     

Evidence for short-time divergence and long-time conservation of tissue-specific expression after gene duplication

Gene duplication is one of the main mechanisms by which genomes can acquire novel functions. It has been proposed that the retention of gene duplicates can be associated to processes of tissue expression divergence. These models predict that acquisition of divergent expression patterns should be acquired shortly after the duplication, and that larger divergence in tissue expression would be expected for paralogs, as compared to orthologs of a similar age. Many studies have shown that gene duplicates tend to have divergent expression patterns and that gene family expansions are associated with high levels of tissue specificity. However, the timeframe in which these processes occur have rarely been investigated in detail, particularly in vertebrates, and most analyses do not include direct comparisons of orthologs as a baseline for the expected levels of tissue specificity in absence of duplications. To assess the specific contribution of duplications to expression divergence, we combine here phylogenetic analyses and expression data from human and mouse. In particular, we study differences in spatial expression among human-mouse paralogs, specifically duplicated after the radiation of mammals, and compare them to pairs of orthologs in the same species. Our results show that gene duplication leads to increased levels of tissue specificity and that this tends to occur promptly after the duplication event.     

Time course and localization of DNA synthesis during wound healing of potato tuber tissue

DNA synthesis is induced in potato tuber tissue by wounding and starts after a lag period of about 8 hr. As demonstrated by the incorporation of labeled precursors, it reaches its peak between 14 and 18 hr after cutting, and returns to the initial low level before 24 hr, the time of first cell divisions. DNA synthesis is confined to those 2 or 3 cell layers below the wound, where cell division and starch degradation are observed later. Protein synthesis and increase in respiration extend much deeper into the tissue. Both the time course of DNA synthesis and its spatial distribution show patterns different from those of other wound-induced metabolic activities. As wound healing in potato tuber tissue involves the establishment of specific patterns with respect to the time course of induced metabolic activities as well as their spatial distribution, it should be considered and studied as a developmental process. The practical advantages of the system are discussed.     

Adenovirus type 12-induced rat tumor cells of neuroepithelial origin: persistence and expression of the viral genome.

Four cell lines derived from adenovirus type 12-induced rat brain tumors were studied. The polyploid cells displayed neuroepithelial characteristics and were transplantable into syngeneic rats and nude mice. In tissue culture the cells grew in monolayers and multilayers. A very high saturation density was reached, and the cells plated in agar and were easily agglutinated with low concentrations of concanavalin A. Between 2 and 11 copies of the viral genome per diploid cellular genome were detected by reassociation kinetics analysis in the different lines. The patterns of distribution of viral DNA sequences in these lines, as revealed by blot analysis, suggest colinear integration of the intact viral genome into the cellular DNA. The patterns of integration were stable after more than 15 months of prolonged tissue culture and after animal reimplantation. Integration patterns were identical in three of the tumor lines and different in another line. Viral sequences were transcribed. The extent of homology found toward adenovirus type 12 DNA in polyadenylated polysome-associated mRNA isolated from the tumor lines suggests that the early and some of the late genes of adenovirus type 12 DNA are transcribed in these tumor cells. Infectious virus was not rescuable from these lines.     

Comparison of isozyme patterns between Spirometra erinacei and Spirometra mansonoides by isoelectric focusing.

No differences were observed in the isozyme patterns of 4 enzymes examined between fresh samples stored at -80 C and samples stored at room temperature for 10 days after lyophilization, which supports the validity of comparing lyophilized samples to fresh frozen tissue. Mature proglottids as well as plerocercoids of Spirometra erinacei from Japan and Australia were indistinguishable by comparison of isozyme patterns after isoelectric focusing. The isozyme patterns of acid phosphatase, glucosephosphate isomerase (GPI), and mannosephosphate isomerase from plerocercoids of Spirometra mansonoides were distinctly different from those of plerocercoids of S. erinacei. The adenylate kinase isozyme patterns of the mature proglottids of S. mansonoides were also distinctly different from those of the mature proglottids and the plerocercoids of S. erinacei. The GPI isozyme pattern of the mature proglottids of S. mansonoides was also distinguishable from the GPI patterns of those of S. erinacei. These electrophoretic data suggest that the S. erinacei from Japan and Australia are closely related, if not identical, but that S. mansonoides is genetically distinct from S. erinacei.     

Anatomical site of pheromone accumulation and temporal pattern of pheromone emission in the ambrosia beetle Megaplatypus mutatus

Megaplatypus mutatus (Chapuis) is a native South American ambrosia beetle that attacks live hardwood trees (e.g. Populus spp.), causing important economic losses to commercial plantations. Male beetles release the main components of the sex pheromone, namely (+)‐6‐methyl‐5‐hepten‐2‐ol [(+)‐sulcatol, or retusol] and 6‐methyl‐5‐hepten‐2‐one (sulcatone), when colonizing suitable hosts. The hindgut is shown to be the anatomical site of pheromone accumulation within males, the enantiomeric composition of sulcatol in this tissue is 99%‐(+) and sulcatol is first detectable in this tissue on days 1–2 after gallery initiation. Peak accumulation of sulcatol occurs on days 5–12 after gallery initiation. Trace quantities of sulcatone are also observed during the same period. Both pheromone components are present in male emissions from three host species (Populus×canadensis, Populus alba and Casuarina stricta) between days 2 and 12 after gallery initiation, although sulcatone is always present in low concentrations. The temporal patterns of sulcatol and sulcatone accumulation or storage in male M. mutatus correspond to the temporal patterns of emission.     

The hantaviral load in tissues of naturally infected rodents

Hantaviruses cause a lifelong and asymptomatic infection in naturally infected hosts as well as in experimentally infected rodents. Understanding the ecology and pathogenesis of hantaviruses requires an interdisciplinary research approach, which links laboratory experiments with results gained from field studies. Although several studies report hantavirus persistence and tissue infection patterns for experimentally infected rodents, field data is very limited. For this reason, the aim of our study was to investigate Puumala, Dobrava and Saaremaa virus RNA loads and tissue infection patterns in their natural reservoirs. Hantavirus RNA was demonstrated in all tested internal organs and blood samples of 14 naturally infected rodent hosts. However, the concentration of a specific virus differs depending on the virus, the host and the organ tested. Above all, the Dobrava virus showed a considerably higher viral load in all internal organs and blood samples of infected Apodemus flavicollis hosts. Results obtained in the study support the thesis that virus RNA load reaches its peak in the first month after infection, presumably after the virus has spread throughout all internal organs. This also implies that recently infected rodents are more important for transmission of the virus in the community.     

Stability of polarity in the epidermis of a beetle, Tenebrio molitor L.

Cell polarity in the insect epidermis may be coupled to the orientation of anisometric cuticle components. Rotation of squares of sternite epidermis in the larva results in a corresponding rotation in the highly ordered orientation of cuticle fibers in the adult “crossply” cuticle. The patterns of fiber orientation resulting from graft rotation can be explained by the presence of an axial gradient of positional information.The polarity of the rotated tissue is, however, not fixed. Interaction between the polarity of the graft and host tissue may result in a partial shift of graft polarity toward the axial polarity of the host tissue. This interaction is apparently restricted to a limited period of the cell cycle: cell division. In Tenebrio, the sternite epidermis proliferates only once during metamorphosis, 140–90 hr before pupation. Rotational grafts performed before, during, and after this period present a graded series of “relaxation” patterns in fiber orientation in the graft area. Maximal graft repolarization coincides with maximal cell division on the sternite. The epidermal gradient, or cell response to the gradient, appears to be nonlinear along the segment.If no cell division intervenes between graft rotation and fiber deposition, graft polarity remains stable. This stability necessitates a “memory” component in the epidermis. It is suggested that periodic assessment of tissue polarity occurs concomitant with a particular process of cell division.     

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4–7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.     

Hypoxically inducible barley alanine aminotransferase: cDNA cloning and expression analysis

A 1.75 kb cDNA containing the entire coding sequence of the hypoxically inducible alanine aminotransferase (AlaAT) from barley roots was isolated and sequenced. This clone has an open reading frame of 1446 bp, and a deduced amino acid sequence of 482 residues, giving an estimated protein molecular mass of 52 885 Da. RNA blot analysis of barley root tissue showed a 4-fold increase of a single AlaAT-2 mRNA band after 12–24 hours of hypoxic stress, followed by a decrease in message levels after 48 h of hypoxic conditions. AlaAT-2 protein concentration increased in a similar pattern to AlaAT activity in root tissue, to almost 6-fold the aerobic level after 96 h of hypoxic stress. AlaAT-2 activity increased more than 2-fold in roots of Panicum miliaceum exposed to hypoxia, and is the same isoform as the light inducible AlaAT in P. miliaceum leaves. The unique expression patterns of AlaAT-2 in root and leaf tissue upon exposure to different environmental stimuli is also discussed.     

Dynamical modeling of the cholesterol regulatory pathway with Boolean networks

Background

RNA silencing has been implicated in virus symptom development in plants. One common infection symptom in plants is the formation of chlorotic tissue in leaves. Chlorotic and healthy tissue co-occur on a single leaf and form patterns. It has been shown that virus levels in chlorotic tissue are high, while they are low in healthy tissue. Additionally, the presence of siRNAs is confined to the chlorotic spots and the boundaries between healthy and infected tissue. These results strongly indicate that the interaction between virus growth and RNA silencing plays a role in the formation of infection patterns on leaves. However, how RNA silencing leads to the intricate patterns is not known.

Results

Here we elucidate the mechanisms leading to infection patterns and the conditions which lead to the various patterns observed. We present a modeling approach in which we combine intra- and inter-cellular dynamics of RNA silencing and viral growth. We observe that, due to the spread of viruses and the RNA silencing response, parts of the tissue become infected while other parts remain healthy. As is observed in experiments high virus levels coincide with high levels of siRNAs, and siRNAs are also present in the boundaries between infected and healthy tissue. We study how single- and double-stranded cleavage by Dicer and amplification by RNA-dependent RNA polymerase can affect the patterns formed.

Conclusion

This work shows that RNA silencing and virus growth within a cell, and the local spread of virions and siRNAs between cells can explain the heterogeneous spread of virus in leaf tissue, and therewith the observed infection patterns in plants.     

Hybrid isozymes of pyruvate kinase appear during avian cardiac development

Pyruvate kinase isozyme patterns in the ventricle of developing chicks shift gradually from one dominated by type K at ten days of embryonic development to the adult pattern, which is dominated by type M. Hybrid isozymes are apparent throughout development and are most prominent from two days before hatching until at least 14 days after hatching. These hybrid isozymes indicate simultaneous synthesis of the two subunit types in the same cells.The complex isozyme patterns of the chick heart probably limit the usefulness of simple kinetic analyses on tissue extracts for determing isozymic compositions during development.     

Tissue and Cellular Expression Patterns of Porcine CFTR: Similarities to and Differences From Human CFTR

Emerging porcine models of cystic fibrosis (CF) are expected to mimic the human disease more closely than current mouse models do. However, little is known of the tissue and cellular expression patterns of the porcine CF transmembrane conductance regulator (pCFTR) and possible differences from human CFTR (hCFTR). Here, the expression pattern of pCFTR was systematically established on the mRNA and protein levels. Using specific anti-pCFTR antibodies, the majority of the protein was immunohistochemically detected on paraffin-embedded sections and on cryostate sections in the apical cytosol of intestinal crypt epithelial cells, nasal, tracheal, and bronchial epithelial cells, and other select, mostly glandular epithelial cells. Confocal laser scanning microscopy with co-localization of the Golgi marker 58K localized the protein in the cytosol between the Golgi apparatus and the apical cell membrane with occasional punctate or diffuse staining of the apical membrane. The tissue and cellular distribution patterns were confirmed by RT-PCR from whole tissue lysates or select cells after laser capture microdissection. Thus, expression of pCFTR was found to largely resemble that of hCFTR except for the kidney, brain, and cutaneous glands, which lack expression in pigs. Species-specific differences between pCFTR and hCFTR may become relevant for future interpretations of the CF phenotype in pig models. (J Histochem Cytochem 58:785–797, 2010)     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.