The aberrant,a morphological mutant of Hydra attenuata,has altered inhibition properties

Transplantation experiments have led to the idea that developmental gradients are involved in the patterning processes of hydra. Head activation and head inhibition gradients appear to be involved in head formation. More could be learned about pattern formation if these gradients could be altered in the animal. One approach to this end is to study mutants suspected of having altered gradients. Such mutants may be identified by their altered morphology. In this investigation the aberrant, a morphological mutant of Hydra attenuata, was examined for alterations in its gradients. The aberrant was found to have changes in its inhibition and polarity properties. The inhibition gradient was steeper than normal and the level of inhibition was higher than normal near the head. In addition, the amount of inhibition transmitted from the head was greater than normal, while the amount of inhibition transmitted through the gastric region was less than normal. The polarity of the aberrant gastric region was more difficult than normal to reverse, and the aberrant extremities caused faster polarity reversal than normal extremities. The significance of these changes is discussed in relation to pattern formation.     

Nematocyte development in Hydra attenuata is dependent on both the interstitial cells and the epithelial cells

The aberrant, a morphological mutant of Hydra attenuata, has altered patterns of the development and distribution of nematocytes. The number of nematoblasts and nematocytes is higher in the aberrant than in the normal. Stenotele differentiation is incomplete and the numbers of desmonemes and holotrichous isohrizas mounted on the body column are much higher than normal. Because nematocytes arise by differentiation from the interstitial cells, epithelial cell/interstitial cell chimeras between the aberrant and normal strains were made to determine whether the lesion giving rise to the alterations in the mutant was due to the epithelial cells or a cell type in the nematocyte lineage. Only the chimera in which both cell types were derived from the aberrant exhibited the altered nematocyte development. If the chimera contained a normal cell type, either epithelial cell or interstitial cell, nematocyte development was normal. Thus, both epithelial cells and cells of the nematocyte lineage are involved in the control of nematocyte development. A defect in one of the lineages can be compensated for by the other cell type.     

Axial Patterning in Hydra

Morphogen gradients play an important role in pattern formation during early stages of embryonic development in many bilaterians. In an adult hydra, axial patterning processes are constantly active because of the tissue dynamics in the adult. These processes include an organizer region in the head, which continuously produces and transmits two signals that are distributed in gradients down the body column. One signal sets up and maintains the head activation gradient, which is a morphogenetic gradient. This gradient confers the capacity of head formation on tissue of the body column, which takes place during bud formation, hydra''s mode of asexual reproduction, as well as during head regeneration following bisection of the animal anywhere along the body column. The other signal sets up the head inhibition gradient, which prevents head formation, thereby restricting bud formation to the lower part of the body column in an adult hydra. Little is known about the molecular basis of the two gradients. In contrast, the canonical Wnt pathway plays a central role in setting up and maintaining the head organizer.Morphogen gradients play a critical role in the early stages of embryogenesis in a number of metazoans in that they initiate and are involved in axial patterning processes. Such a gradient also plays a role in axial patterning in hydra, a primitive metazoan. However, unlike in most metazoans, this gradient is continuously active in an adult hydra as part of the tissue dynamics of the adult animal.The structure of a hydra is fairly simple (Fig. ​(Fig.1).1). It consists of a single axis with radial symmetry, which contains a head, body column, and foot along the axis. The head consist of two parts: the hypostome in the apex, and the tentacle zone from which the tentacles emerge in the basal part of the head. The body column has three parts: the gastric region and peduncle in the apical, and basal parts with a budding zone between the gastric region and peduncle. Buds, hydra''s mode of asexual reproduction, emerge from the budding zone between the gastric region and peduncle.Open in a separate windowFigure 1.Longitudinal cross section of an adult hydra. The multiple regions are labeled. The two protrusions from the body column are early and late stages of bud development. The arrows indicate the direction of tissue displacement. (Reprinted from Bode 2001.)Three cell lineages are involved. The axis consists of a cylindrical shell that is made up of two concentric epithelial layers, the ectoderm and endoderm, which are separated by a basement membrane. Interspersed among the epithelial cells of both layers are the cells of the third lineage, the interstitial cell lineage. It consists of interstitial cells, which are multipotent stem cells (David and Murphy 1977), located primarily in the ectoderm throughout the body column. They give rise to neurons, secretory cells, and nematocytes, which are the stinging cells that are typical of cnidarians, as well as gametes when a hydra undergoes sexual reproduction (David and Murphy 1977).In an adult hydra, the epithelial cells of both layers are constantly in the mitotic cycle (David and Campbell 1972; Campbell and David 1974). The expanding tissue in the upper part of the body column is continuously displaced apically into the head (Fig. 1). Once there, it is displaced onto and along the tentacles or into the hypostome, and eventually sloughed when reaching the extremities (Campbell 1967; Otto and Campbell 1977). Tissue in the remainder of the body column is displaced basally either onto developing buds, or further down onto the foot, where it is sloughed at the bottom of the foot. Thus, the tissues of an adult hydra are continuously in a steady state of production and loss. As a hydra has no defined lifetime (Martinez 1998), this activity goes on indefinitely.     

Uterine epithelial cell regulation of DC-SIGN expression inhibits transmitted/founder HIV-1 trans infection by immature dendritic cells

Background

Sexual transmission accounts for the majority of HIV-1 infections. In over 75% of cases, infection is initiated by a single variant (transmitted/founder virus). However, the determinants of virus selection during transmission are unknown. Host cell-cell interactions in the mucosa may be critical in regulating susceptibility to infection. We hypothesized in this study that specific immune modulators secreted by uterine epithelial cells modulate susceptibility of dendritic cells (DC) to infection with HIV-1.

Methodology/Principal Findings

Here we report that uterine epithelial cell secretions (i.e. conditioned medium, CM) decreased DC-SIGN expression on immature dendritic cells via a transforming growth factor beta (TGF-β) mechanism. Further, CM inhibited dendritic cell-mediated trans infection of HIV-1 expressing envelope proteins of prototypic reference. Similarly, CM inhibited trans infection of HIV-1 constructs expressing envelopes of transmitted/founder viruses, variants that are selected during sexual transmission. In contrast, whereas recombinant TGF- β1 inhibited trans infection of prototypic reference HIV-1 by dendritic cells, TGF-β1 had a minimal effect on trans infection of transmitted/founder variants irrespective of the reporter system used to measure trans infection.

Conclusions/Significance

Our results provide the first direct evidence for uterine epithelial cell regulation of dendritic cell transmission of infection with reference and transmitted/founder HIV-1 variants. These findings have immediate implications for designing strategies to prevent sexual transmission of HIV-1.     

The role of bronchial epithelial cells in the pathogenesis of COPD in Z-alpha-1 antitrypsin deficiency

Background

Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown.We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients.

Methods

Experiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of < 0.05 was considered to be significant.

Results

Alpha-1 antitrypsin polymers were found at a higher concentration in the culture medium of ex vivo bronchial epithelial cells from Z-variant homozygotes, compared with M-variant homozygotes (P < 0.01), and detected in the bronchial epithelial cells and submucosa of patient biopsies. Oncostatin M significantly increased the expression of alpha-1 antitrypsin mRNA and protein (P < 0.05), and the presence of Z-variant polymers in ex vivo cells (P < 0.01).

Conclusions

Polymers of Z-alpha-1 antitrypsin form in bronchial epithelial cells, suggesting that these cells may be involved in the pathogenesis of lung emphysema and in bronchial epithelial cell dysfunction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0112-3) contains supplementary material, which is available to authorized users.     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

Forming Patterns in Development without Morphogen Gradients: Scattered Differentiation and Sorting Out

Few mechanisms provide alternatives to morphogen gradients for producing spatial patterns of cells in development. One possibility is based on the sorting out of cells that initially differentiate in a salt and pepper mixture and then physically move to create coherent tissues. Here, we describe the evidence suggesting this is the major mode of patterning in Dictyostelium. In addition, we discuss whether convergent evolution could have produced a conceptually similar mechanism in other organisms.A limited number of processes are thought to regulate the differentiation of specialized cell types and their organization to form larger scale structures, such as organs or limbs, during embryonic development. First, early embryological experiments revealed a patterning process that depends on special “organizing” regions in the embryo. This idea was encapsulated as “positional information” and led to the concept of morphogen gradients (Fig. 1) (Wolpert 1996). In addition, cytoplasmic determinants have been shown to direct development along different lines when they are partitioned unequally between daughter cells by asymmetric cell division (Betschinger and Knoblich 2004). Finally, short-range inductive signaling can specify cells at a local level and when reiterated produces highly ordered structures (Simpson 1990; Freeman 1997; Meinhardt and Gierer 2000).Open in a separate windowFigure 1.Alternative ways of patterning cells during development. (A) Patterning by “positional information”: A group of undifferentiated cells is patterned by a morphogen diffusing from a pre-established source, producing a concentration gradient. Cells respond according to the local morphogen concentration, becoming red, white, or blue. (B, C) Patterning without positional information: This is a two-step process in which different cell types first differentiate mixed up with each other, and then sort out. The initial differentiation can be controlled by strictly local interactions between the cells, as in lateral inhibition (B), or by a global signal to which cells respond with different sensitivities and whose concentration they regulate by negative feedback (C). Once sorting has occurred, the global inducer forms a reverse gradient, which could then convey positional information for further patterning events.The question then arises of whether evolution has devised any further global patterning mechanisms. One possibility that has been repeatedly considered, but not firmly established as a general mechanism, is based on sorting out. In this process, pattern is produced in two steps: (1) Different cell types are initially specified from a precursor pool independent of their position to produce a salt and pepper mixture and (2) the mixture of cell types is resolved into discrete tissues by the physical movement and sorting out of the cells (Fig. 1). Consequently, this mechanism does not involve positional information. However, it can actually provide the conditions under which positional signaling and morphogen gradients can arise, if the resolved tissues then act as sources and sinks for signal molecules.We first describe the powerful evidence that this alternative patterning process is used during the developmental cycle of the social amoeba Dictyostelium discoideum, and then consider the possibility that this patterning strategy may be used more widely.     

The Moraxella catarrhalis-induced pro-inflammatory immune response is enhanced by the activation of the epidermal growth factor receptor in human pulmonary epithelial cells

Background

Chronic lower airway inflammation is considered to be a major cause of pathogenesis and disease progression in chronic obstructive pulmonary disease (COPD). Moraxella catarrhalis is a COPD-associated pathogen causing exacerbations and bacterial colonization in the lower airways of patients, which may contribute to chronic inflammation. Increasing evidence suggests that the epidermal growth factor receptor (EGFR) modulates inflammatory processes in the human airways. The goal of this study was to investigate the role of EGFR in the M. catarrhalis-induced pro-inflammatory immune response in airway epithelial cells.

Methods

The effects of inhibition and gene silencing of EGFR on M. catarrhalis-dependent pro-inflammatory cytokine expression in human primary bronchial epithelial cells (NHBEs), as well as the pulmonary epithelial cell lines BEAS-2B and A549 were analyzed. We also assessed the involvement of EGFR-dependent ERK and NF-κB signaling pathways.

Results

The M. catarrhalis-induced pro-inflammatory immune response depends, at least in part, on the phosphorylation and activation of the EGF receptor. Interaction of M. catarrhalis with EGFR increases the secretion of pro-inflammatory cytokines, which is mediated via ERK and NF-κB activation.

Conclusion

The interaction between M. catarrhalis and EGFR increases airway inflammation caused by this pathogen. Our data suggest that the inhibition of EGFR signaling in COPD could be an interesting target for reducing M. catarrhalis-induced airway inflammation.     

A Novel Cell Death Gene Acts to Repair Patterning Defects in Drosophila melanogaster

Cell death is a mechanism utilized by organisms to eliminate excess cells during development. Here, we describe a novel regulator of caspase-independent cell death, Mabiki (Mabi), that is involved in the repair of the head patterning defects caused by extra copies of bicoid in Drosophila melanogaster. Mabiki functions together with caspase-dependent cell death mechanisms to provide robustness during development.     

Irregular xylem 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis

Large quantities of mucilage are synthesized in seed coat epidermis cells during seed coat differentiation. This process is an ideal model system for the study of plant cell wall biosynthesis and modifications. In this study, we show that mutation in Irregular Xylem 7 (IRX7) results in a defect in mucilage adherence due to reduced xylan biosynthesis. IRX7 was expressed in the seeds from 4 days post-anthesis (DPA) to 13 DPA, with the peak of expression at 13 DPA. The seed coat epidermis cells of irx7 displayed no aberrant morphology during differentiation, and these cells synthesized and deposited the same amount of mucilage as did wild type (WT) cells. However, the distribution of the water-soluble vs. adherent mucilage layers was significantly altered in irx7 compared to the WT. Both the amount of xylose and the extent of glycosyl linkages of xylan was dramatically decreased in irx7 water-soluble and adherent mucilage compared to the WT. The polymeric structure of water-soluble mucilage was altered in irx7, with a total loss of the higher molecular weight polymer components present in the WT. Correspondingly, whole-seed immunolabeling assays and dot-immunoassays of extracted mucilage indicated dramatic changes in rhamnogalacturonan I (RG I) and xylan epitopes in irx7 mucilage. Furthermore, the crystalline cellulose content was significantly reduced in irx7 mucilage. Taken together, these results indicate that xylan synthesized by IRX7 plays an essential role in maintaining the adhesive property of seed coat mucilage, and its structural role is potentially implemented through its interaction with cellulose.     

Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells

Background

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.

Methodology/Principal Findings

Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta''), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.

Conclusions

alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.     

Nitric oxide and airway epithelial barrier function: Regulation of tight junction proteins and epithelial permeability

Acute airway inflammation is associated with enhanced production of nitric oxide (NO) and altered airway epithelial barrier function, suggesting a role of NO or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o− cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2−/− mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H₂O₂, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity.     

An analysis of segmentation dynamics throughout embryogenesis in the centipede <Emphasis Type="Italic">Strigamia maritima</Emphasis>

Background

Most segmented animals add segments sequentially as the animal grows. In vertebrates, segment patterning depends on oscillations of gene expression coordinated as travelling waves in the posterior, unsegmented mesoderm. Recently, waves of segmentation gene expression have been clearly documented in insects. However, it remains unclear whether cyclic gene activity is widespread across arthropods, and possibly ancestral among segmented animals. Previous studies have suggested that a segmentation oscillator may exist in Strigamia, an arthropod only distantly related to insects, but further evidence is needed to document this.

Results

Using the genes even skipped and Delta as representative of genes involved in segment patterning in insects and in vertebrates, respectively, we have carried out a detailed analysis of the spatio-temporal dynamics of gene expression throughout the process of segment patterning in Strigamia. We show that a segmentation clock is involved in segment formation: most segments are generated by cycles of dynamic gene activity that generate a pattern of double segment periodicity, which is only later resolved to the definitive single segment pattern. However, not all segments are generated by this process. The most posterior segments are added individually from a localized sub-terminal area of the embryo, without prior pair-rule patterning.

Conclusions

Our data suggest that dynamic patterning of gene expression may be widespread among the arthropods, but that a single network of segmentation genes can generate either oscillatory behavior at pair-rule periodicity or direct single segment patterning, at different stages of embryogenesis.

     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

In Vitro Effects of a Small-Molecule Antagonist of the Tcf/?-Catenin Complex on Endometrial and Endometriotic Cells of Patients with Endometriosis

Background

Our previous studies suggested that aberrant activation of Wnt/ß-catenin signaling might be involved in the pathophysiology of endometriosis. We hypothesized that inhibition of Wnt/ß-catenin signaling might result in inhibition of cell proliferation, migration, and/or invasion of endometrial and endometriotic epithelial and stromal cells of patients with endometriosis.

Objectives

The aim of the present study was to evaluate the effects of a small-molecule antagonist of the Tcf/ß-catenin complex (PKF 115–584) on cell proliferation, migration, and invasion of endometrial and endometriotic epithelial and stromal cells.

Methods

One hundred twenty-six patients (78 with and 48 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of PKF 115–584 on cell proliferation, migration, and invasion and on the Tcf/ß-catenin target genes were evaluated in endometrial epithelial and stromal cells of patients with and without endometriosis, and in endometrial and endometriotic epithelial and stromal cells of the same patients.

Results

The inhibitory effects of PKF 115–584 on cell migration and invasion in endometrial epithelial and stromal cells of patients with endometriosis prepared from the menstrual phase were significantly higher than those of patients without endometriosis. Levels of total and active forms of MMP-9 were significantly higher in epithelial and stromal cells prepared from menstrual endometrium in patients with endometriosis compared to patients without endometriosis. Treatment with PKF 115–584 inhibited MMP-9 activity to undetectable levels in both menstrual endometrial epithelial and stromal cells of patients with endometriosis. The number of invasive cells was significantly higher in epithelial and stromal cells of endometriotic tissue compared with matched eutopic endometrium of the same patients. Treatment with PKF 115–584 decreased the number of invasive endometriotic epithelial cells by 73% and stromal cells by 75%.

Conclusions

The present findings demonstrated that cellular mechanisms known to be involved in endometriotic lesion development are inhibited by targeting the Wnt/β-catenin pathway.