Equivalent genetic regulatory networks in different contexts recover contrasting spatial cell patterns that resemble those in Arabidopsis root and leaf epidermis: a dynamic model

In Arabidopsis thaliana, leaf and root epidermis hairs exhibit contrasting spatial arrangements even though the genetic networks regulating their respective cell-fate determination have very similar structures and components. We integrated available experimental data for leaf and root hair patterning in dynamic network models which may be reduced to activator-inhibitor models. This integration yielded expected results for these kinds of dynamic models, including striped and dotted cell patterns which are characteristic of root and leaf epidermis, respectively. However, these formal tools have led us to novel insights on current data and to put forward precise hypotheses which can be addressed experimentally. In particular, despite subtle differences in the root and leaf networks, these have equivalent dynamical behaviors. Our simulations also suggest that only when a biasing signal positively affects an activator in the network, the system recovers striped cellular patterns similar to those of root epidermis. We also postulate that cell shape may affect pattern stability in the root. Our results thus support the idea that in this and other cases, contrasting spatial cell patterns and other evolutionary morphogenetic novelties originate from conserved genetic network modules subject to divergent contextual traits.     

New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.     

Microfluidic formation of single cell array for parallel analysis of Ca release-activated Ca (CRAC) channel activation and inhibition

High-throughput single cell analysis is required for understanding and predicting the complex stochastic responses of individual cells in changing environments. We have designed a microfluidic device consisting of parallel, independent channels with cell-docking structures for the formation of an array of individual cells. The microfluidic cell array was used to quantify single cell responses and the distribution of response patterns of calcium channels among a population of individual cells. In this device, 15 cell-docking units in each channel were fabricated with each unit containing 5 sandbag structures, such that an array of individual cells was formed in 8 independent channels. Single cell responses to different treatments in different channels were monitored in parallel to study the effects of the specific activator and inhibitor of the Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Multichannel detection was performed to obtain the response patterns of the population of cells within this single cell array. The results demonstrate that it is possible to acquire single cell features in multichannels simultaneously with passive structural control, which provides an opportunity for high-throughput single cell response analysis in a microfluidic chip.     

Kolmogorov complexity of epithelial pattern formation: The role of regulatory network configuration

The tissues of multicellular organisms are made of differentiated cells arranged in organized patterns. This organization emerges during development from the coupling of dynamic intra- and intercellular regulatory networks. This work applies the methods of information theory to understand how regulatory network structure both within and between cells relates to the complexity of spatial patterns that emerge as a consequence of network operation. A computational study was performed in which undifferentiated cells were arranged in a two dimensional lattice, with gene expression in each cell regulated by identical intracellular randomly generated Boolean networks. Cell–cell contact signalling between embryonic cells is modeled as coupling among intracellular networks so that gene expression in one cell can influence the expression of genes in adjacent cells. In this system, the initially identical cells differentiate and form patterns of different cell types. The complexity of network structure, temporal dynamics and spatial organization is quantified through the Kolmogorov-based measures of normalized compression distance and set complexity. Results over sets of random networks that operate in the ordered, critical and chaotic domains demonstrate that: (1) ordered and critical networks tend to create the most information-rich patterns; (2) signalling configurations in which cell-to-cell communication is non-directional mostly produce simple patterns irrespective of the internal network domain; and (3) directional signalling configurations, similar to those that function in planar cell polarity, produce the most complex patterns, but only when the intracellular networks function in non-chaotic domains.     

鹅掌楸油细胞的发育解剖学研究

鹅掌楸油细胞比相邻组织细胞分化,因而在叶肉细胞未完全分化的叶原基、幼叶以及未完全分化的幼茎中,都可看到已分化的油细胞。通常,在第二叶原基中可发现油细胞原始细胞,由于其染色深、细胞核大而易与周围组织的细胞相区别。以后,油细胞逐步液泡化,直至形成一个大的液泡,此时,细胞核呈扁平状,并与细胞质一起成为一薄层围绕着大液泡。当油细胞发育成熟后,细胞质及核开始解体,整个油细胞的腔由大液泡充满,成为油囊,在部分     

Why do lupin cell cultures fail to produce alkaloids in large quantities?

Cell suspension cultures of lupins and other legumes accumulate alkaloids at levels that are 2 to 3 orders of magnitude lower as compared to the alkaloid concentrations in leaves of the respective differentiated plants. In the plant the alkaloids are formed in leaf chloroplasts and are then translocated in the phloem sap to the other plant organs, where they are preferentially stored in epidermal tissues. In the cell cultures the formation of specialized storage tissues is probably repressed. As a consequence the alkaloids formed cannot be stored in large quantities. They are degraded rapidly either in the cells or in the cell culture medium, which contains a number of hydrolytic and oxidative exoenzymes.     

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.     

Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and β-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and β-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, β-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas β-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the β-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and β-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and β-III-tubulin during the neural differentiation of H9 cells.     

Conversion of ES cells to columnar epithelia by hensin and to squamous epithelia by laminin

Single-layered epithelia are the first differentiated cell types to develop in the embryo, with columnar and squamous types appearing immediately after blastocyst implantation. Here, we show that mouse embryonic stem cells seeded on hensin or laminin, but not fibronectin or collagen type IV, formed hemispheric epithelial structures whose outermost layer terminally differentiated to an epithelium that resembled the visceral endoderm. Hensin induced columnar epithelia, whereas laminin formed squamous epithelia. At the egg cylinder stage, the distal visceral endoderm is columnar, and these cells begin to migrate anteriorly to create the anterior visceral endoderm, which assumes a squamous shape. Hensin expression coincided with the dynamic appearance and disappearance of columnar cells at the egg cylinder stage of the embryo. These expression patterns, and the fact that hensin null embryos (and those already reported for laminin) die at the onset of egg cylinder formation, support the view that hensin and laminin are required for terminal differentiation of columnar and squamous epithelial phenotypes during early embryogenesis.     

Analysis of the local organization and dynamics of cellular actin networks

A ctin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.     

A local activator-inhibitor model of vertebrate skin patterns

A model for vertebrate skin patterns is presented in which the differentiated (colored) pigment cells produce two diffusible morphogens, an activator and an inhibitor. The concentrations of these two substances at any point on the skin determine whether a pigment cell at that point will be colored or not. Computer simulations with this model show many realistic features of spot and stripe patterns found in vertebrates.     

Clonal sublines of rat neurotumor RT4 and cell differentiation: II. A conversion coupling of tumorigenicity and a glial property

RT4 is a neurotumor induced by ethylnitrosourea injection of a newborn BDIX rat. We demonstrated previously that heterogeneity in early cultures of RT4 tumor cells can be regularly reproduced in cultures of clonal stem cells (cell type conversion). Our previous studies included morphology, differentiation of neural properties, and chromosome number of “tumor-derived” and “stem cell-derived” differentiated cells. In this paper, these two sets of differentiated cells were examined further for three additional parameters, all of which are related to malignancy. The stem cell (AC) and one type of differentiated cell (D) cause tumors when subcutaneously injected into syngeneic animals, while the other two types (B and E) do not. The amounts of a 250,000 molecular weight cell surface protein, which is probably equivalent to LETS protein (large external transformation-sensitive protein) of hamster and mouse, and the levels of plasminogen activator were examined as possible markers of malignancy. As anticipated, nontumorigenic cells generally have a large amount of the 250,000 molecular weight cell surface protein and are low in plasminogen activator activities, whereas the reverse is true for tumorigenic cells. This supports the idea that B and E cells are nontumorigenic revertants. The cell type conversion phenomenon of RT4 neurotumor and the differentiation of mouse teratoma and myeloid leukemic cells share a number of similarities, but differ in that differentiated RT4 cells can propagate in vitro even after loss of tumorigenicity. The concomitant expression of tumorigenicity and the S100 protein production of the D cell, or of nontumorigenicity and B and E cell differentiation upon the conversion of the stem cell, may suggest a regulational coupling between the tumorigenicity and the expression of a glial protein (S100 protein) in D cells.     

青藏高原草地植物叶解剖特征

运用常规石蜡制片技术对我国青藏高原66种草地植物优势种的叶解剖特征进行研究,并分析了叶解剖特征与海拔、生长季降水及生长季均温之间的关系.结果表明:青藏高原草地植物叶片具有很多适应高寒环境的结构特征,如表皮层厚且表皮细胞大小差异显著,表皮毛等表皮附属物发达,异细胞丰富,通气组织普遍发达等;叶片各组成部分厚度的变异程度不同,其中海绵组织厚度变异最大,其次为上角质层、下表皮层、下角质层、上表皮层、栅栏组织,叶片厚度的变异最小;青藏高原草地植物叶片各组成部分的厚度存在协同进化,上下角质层厚度呈强烈正相关,海绵组织厚度与叶片厚度相关性最强;青藏高原草地植物叶片各组成部分的厚度与海拔、生长季降水、生长季均温3个重要环境变量呈较弱的相关性,总体表现为随海拔升高叶片各组成部分的厚度减小,而随生长季降水和生长季均温的增加叶片厚度增加.     

Extracellular Matrix Components Regulating Glandular Differentiation and the Formation of Basal Lamina of a Human Pancreatic Cancer Cell Line in Vitro

The interaction between the extracellular matrix and human tumor-cell clones S2-013 and S2-020, derived from a pancreatic cancer cell line (SUIT-2), was examined in vitro, using various cell differentiation-promoting matrices in two- and three-dimensional cultures. S2-013 cells (well-differentiated tubular adenocarcinoma in xenografts in nude mice) cultured in Matrigel formed glandular structures. Ultrastructural observation revealed a morphological polarity of cells and a distinct basal lamina. On the other hand, S2-020 cells (poorly differentiated tubular adenocarcinoma in xenografts) cultured in Matrigel formed neither glandular structures nor a basal lamina, but only cell aggregates. The morphology of these two sublines cultured in Matrigel expressed the histological degree of differentiation which they presented in nude mice. In contrast, in type I collagen gel, S2-013 cells formed glandular structures without a basal lamina, and in soft agar, they were able to form neither glandular structures nor a basal lamina. S2-020 cells cultured in type I collagen gel or soft agar formed the same simple cell aggregates as in Matrigel. Matrices used in a three-dimensional culture influenced the degree of differentiation in S2-013 cells but had no effect on the morphological differentiation in S2-020 cells. To detect the factors which induce basal lamina formation, S2-013 cells were cultured on a microporous membrane coated with extracellular matrix components such as laminin, type IV collagen, and fibronectin. S2-013 cells formed a basal lamina only on the laminin. These cell lines may be useful in investigating the mechanisms regulating the formation of glandular structures and basal lamina.     

Relationship of the Electrophysiological Property of Tobacco Cultured Cells to Their Regeneration Ability

The electrophysiological properties of the high-density fractionof tobacco cultured cells (cell clusters) which formed leafprimordia in high yield in Linsmaier-Skoog's medium containingkinetin were compared with those of low-density cell fractionsin which leaf primordia hardly differentiated. Cells of theformer fraction had a higher membrane potential and an electrogenicmechanism which responded to sugars and amino acids. (Received February 12, 1983; Accepted March 23, 1984)     

Heterogeneity in cellular antigen retention structures

The mechanism of presentation of foreign antigens to helper T lymphocytes and the nature of the structures involved in this process are not totally understood. It is well documented that this event is carried out by antigen-presenting cells (APC) (e.g., macrophages, dendritic cells, and B lymphocytes) that internalize the antigen, process it, reexpress it on their membrane surface, and present it to the T cell in the context of major histocompatibility complex class II (Ia) molecules. Recent evidence supports the hypothesis that peptide antigens associate directly with Ia molecules on the APC surface membrane. However, the characteristics of other APC membrane structures potentially involved in antigen presentation are not entirely clear. Previous studies in our laboratories identified a guinea pig macrophage membrane-bound, non-Ia-containing antigenic complex (peak A) formed upon incubation of APC with the octapeptide antigen angiotensin (AII). This complex was capable of stimulating AII-immune guinea pig T cells and thus appeared to contain the immunologically relevant form of the antigen. For this reason it was important to establish whether such complex formation with peptides occurs with other cell types and with other peptide antigens. In the present study we found that other types of cells are also capable of forming such a membrane complex with antigen (peak A) and that this event is not unique to AII. Two other peptides, alpha-melanocyte-stimulating hormone and human fibrinopeptide B, both of which are antigenic in mice, were found to form peak A with a number of murine cell lines. As in our earlier studies with guinea pig macrophages, there was no evidence from these experiments for a role for major histocompatibility complex Ia antigens in the peptide binding observed. Differences in both the amount of peak A formation and the pattern of peptide antigen degradation were found from cell line to cell line for a given peptide, and from peptide to peptide for a given cell line, suggesting cellular heterogeneity in peptide processing and retention. In addition, cross-inhibition studies indicated that there was peptide specificity in the formation of peak A perhaps suggestive of molecular heterogeneity in the structure of peak A. These results indicate that there may be several types of cell surface molecules that specifically bind and retain peptide antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Critical conditions for pattern formation and in vitro tubulogenesis driven by cellular traction fields

In vitro angiogenesis assays have shown that tubulogenesis of endothelial cells within biogels, like collagen or fibrin gels, only appears for a critical range of experimental parameter values. These experiments have enabled us to develop and validate a theoretical model in which mechanical interactions of endothelial cells with extracellular matrix influence both active cell migration--haptotaxis--and cellular traction forces. Depending on the number of cells, cell motility and biogel rheological properties, various 2D endothelial patterns can be generated, from non-connected stripe patterns to fully connected networks, which mimic the spatial organization of capillary structures. The model quantitatively and qualitatively reproduces the range of critical values of cell densities and fibrin concentrations for which these cell networks are experimentally observed. We illustrate how cell motility is associated to the self-enhancement of the local traction fields exerted within the biogel in order to produce a pre-patterning of this matrix and subsequent formation of tubular structures, above critical thresholds corresponding to bifurcation points of the mathematical model. The dynamics of this morphogenetic process is discussed in the light of videomicroscopy time lapse sequences of endothelial cells (EAhy926 line) in fibrin gels. Our modeling approach also explains how the progressive appearance and morphology of the cellular networks are modified by gradients of extracellular matrix thickness.     

Cilia containing 9 ＋ 2 structures grown from immortalized cells

Cilia depend on their highly differentiated structure, a 9 ＋ 2 arrangement, to remove particles from the lung and to transport reproductive cells. Immortalized cells could potentially be of great use in cilia research. Immortalization of cells with cilia structure containing the 9 ＋ 2 arrangement might be able to generate cell lines with such cilia structure. How- ever, whether immortalized cells can retain such a highly differentiated structure remains unclear. Here we demonstrate that （1） using Ela gene transfection, tracheal cells are immortalized; （2） interestingly, in a gel culture the immortalized cells form spherical aggregations within which a lumen is developed; and （3） surprisingly, inside the aggregation, cilia containing a 9 ＋ 2 arrangement grow from the cell＇s apical pole and protrude into the lumen. These results may influence future research in many areas such as understanding the mechanisms of cilia differentiation, cilia generation in other existing cell lines, cilia disorders, generation of other highly differentiated structures besides cilia using the gel culture, immortalization of other ciliated cells with the Ela gene, development of cilia motile function, and establishment of a research model to provide uniform ciliated cells.     

草苁蓉瘤状体的形态解剖学研究

本文详细地观察了草苁蓉瘤状体的形态与结构,结果如下：草苁蓉瘤状体白色或深褐色,大小不等,最大直径约３０ｍｍ幼小时为球形,长大后为夫规则的块状;其解剖学特点是：瘤状体与寄主根连接的部分形成呼器,大量的薄壁细胞和螺纹状管胞伸入木质与根的木质部相连。吸器的真心皮部也由筛管伴胞及薄壁细胞与根的韧皮部相连,两者均缺乏纤维。     

In vivo localization of manganese in the hyperaccumulator Gossia bidwillii (Benth.) N. Snow & Guymer (Myrtaceae) by cryo-SEM/EDAX

Gossia bidwillii (Myrtaceae) is a manganese (Mn)-hyperaccumulating tree native to subtropical eastern Australia. It typically contains foliar Mn levels in excess of 1% dry weight. However, in G. bidwillii and other Mn-hyperaccumulating species, the cellular and subcellular localization of Mn has not been measured. Quantitative in vivo cryo-scanning electron microscopy (SEM)/energy dispersive X-ray analysis (EDAX) was used to localize Mn and other elements in tissue collected from mature trees growing in a natural population. Cryo-SEM showed that the leaf mesophyll is differentiated as a double-layer palisade mesophyll above spongy mesophyll. Transmission electron microscopy (TEM) revealed that the palisade and epidermal cells are highly vacuolated. EDAX data were used to estimate in situ vacuolar Mn concentrations of all cell types in fresh cryo-fixed leaf tissues. The highest average vacuolar Mn concentration of over 500 mM was found in the upper-layer palisade mesophyll, while the lowest concentration of around 100 mM was found in the spongy mesophyll. Qualitative in vivo cryo-SEM/EDAX was employed to further investigate the spatial distribution of Mn in fresh leaf tissues and young bark tissue, which was also found to have a high Mn concentration. It is concluded that Mn distribution in G. bidwillii is quantitatively different to metal distribution in other hyperaccumulating species where the highest localized concentrations of these elements occur in non-photosynthmetic tissues such as epidermal cells and associated dermal structures including trichomes and leaf hairs.