The embryonic expression patterns of zebrafish genes encoding LysM-domains.

The function and structure of LysM-domain containing proteins are very diverse. Although some LysM domains are able to bind peptidoglycan or chitin type carbohydrates in bacteria, in fungi and in plants, the function(s) of vertebrate LysM domains and proteins remains largely unknown. In this study we have identified and annotated the six zebrafish genes of this family, which encode at least ten conceptual LysM-domain containing proteins. Two distinct sub-families called LysMD and OXR were identified and shown to be highly conserved across vertebrates. The detailed characterization of LysMD and OXR gene expression in zebrafish embryos showed that all the members of these sub-families are strongly expressed maternally and zygotically from the earliest stages of a vertebrate embryonic development. Moreover, the analysis of the spatio-temporal expression patterns, by whole mount and fluorescent in situ hybridizations, demonstrates pronounced LysMD and OXR gene expression in the zebrafish brain and nervous system during stages of larval development. None of the zebrafish LysMD or OXR genes was responsive to challenge with bacterial pathogens in embryo models of Salmonella and Mycobacterium infections. In addition, the expression patterns of the OXR genes were mapped in a zebrafish brain atlas.

Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities.

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis.

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severely affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.

Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre-treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.

NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals.

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2-O-methylfucose residue at the reducting-terminal N-acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum). Here we show that transfer of nodZ to Rhizobium leguminosarum blovar (bv) viciae, which produces LCOs that are not modified at the reducing-terminal N-acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N-acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum, Glycine soja, Vigna unguiculate and Leucaena leucocephala. Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.

Concanavalin A-peroxidase labeling in cervical exfoliative cytopathology. I. Labeling of normal squamous cells and the detection of cancer.

The lectin binding capacity of the cell surface of normal flattened exfoliated epithelial cells of the uterine cervix was investigated looking for differences between specimens from normal and cancer patients. The method used was a modified concanavalin A-horseradish peroxidase (Con A-HRP) labeling procedure. Both normal and cancer specimens contain labeled as well as unlabeled usual flattened cells. There is a distinct difference between the labeling intensity of labeled and that of unlabeled cells. Quantification of the labeling results has been achieved using a light microscope equipped with a computerized video system. Apparently healthy persons, having a percentage of labeled flattened cells between 54 and 94% (mean = 73%, SD = 10%, N = 40), were totally discriminated by this method from the cancer patients. These patients with a histologically confirmed squamous cell carcinoma, showed a labeling percentage between 10 and 22% (mean = 15%, SD = 4%, N = 10). Hormonal factors, such as phase of cycle and pill use, appeared to have no significant influence. Statistical analysis revealed that at least 99% of all healthy persons will have a labeling percentage above 45%, while at most 1% of the cancer patients will show a labeling percentage above 30%. When choosing the labeling percentage of 45% as critical value, the Con A-HRP labeling might serve as an additional detection method for cancer of the uterine cervix. Moreover, as it is based on the abundantly present normal cells, and not on the often scarce abnormal cells, the method is not liable to sampling and screening errors.

Tannic acid binding of cell surfaces in normal, premalignant, and malignant squamous epithelium of the human uterine cervix.

Alterations in tannic acid (TA) binding capacity of cell surface carbohydrates in normal, premalignant, and malignant squamous epithelium of the human uterine cervix have been studied using electron microscopic visualization in combination with microdensitometric evaluation. While in normal epithelium there is distinct binding in four to five cell layers of the deep intermediate zone, cells of carcinoma in situ and invasive cancer lesions lack TA binding. In moderate dysplasia an intermediate reacting pattern is found. Deep intermediate cells in areas bordering the carcinoma in situ lesions do not show any binding, although their ultrastructure cannot be distinguished from similar cells in normal tissue. The TA deposition within the deep intermediate zone is probably related to the presence here of glycoprotein-containing membrane-coating granules. The finding that TA binding discriminates between cells in normal squamous epithelium and morphologically normal cells in juxtaposition with lesional areas in premalignant and malignant epithelium opens the possibility for a more reliable cytologic diagnosis of cervical epithelial neoplasia.

Surface pattern differentiation of the epithelial cells of the human uterine ectocervix.

This review considers the SEM cell surface pattern of the human ectocervical stratified squamous epithelium and its differentiation during migration from the basal to the exfoliating layer. The outer surface of flattened superficial cells has been generally characterized by microridges. Although this surface pattern is indeed relatively stable -as follows from an apparently limited sensitivity to preparation factors- it also shows dependency on the hormonal state of the patient. In contrast to the superficial cells very little information is available on the surface structure of the normal intermediate, parabasal and basal cells. The deep surfaces of the superficial cell layers have been examined using a stripping technique, whereas the surfaces of the deeper cell layers have been investigated with a freeze-fracturing technique. During migration from the basal cell layer upwards, the following structural parameters change gradually: the shape of the cells, the size of the intercellular space and the length and width of the cellular processes. As a result of the differentiation process the flattened cells of the superficial zone acquire microridges and crests on the luminal side. These interdigitate respectively, with globular microvilli and furrows present on the basal side of the overlying cell. Crests and furrows delineate regions on the cell surface in which variable microridge- and microvillus-patterns appear. Because crests and furrows are not present on the cell margins but also more centrally on the surface, the cell surface shown "regional" differences. The functional significance of the cell surface structure is discussed. Whereas the surface processes of the basal and parabasal cells presumably are engaged in the enhancement of intercellular contact and must permit metabolic interchange with the intercellular space, the interdigitating processes of superficial cells probably deal with an increase of the surface adhesiveness and in particular the resistance to sideways movements.