Dataset Information

Metal-Nano-Ink Coating for Monitoring and Quantification of Cotyledon Epidermal Cell Morphogenesis.

ABSTRACT: During cotyledon growth, the pavement cells, which make up most of the epidermal layer, undergo dynamic morphological changes from simple to jigsaw puzzle-like shapes in most dicotyledonous plants. Morphological analysis of cell shapes generally involves the segmentation of cells from input images followed by the extraction of shape descriptors that can be used to assess cell shape. Traditionally, replica and fluorescent labeling methods have been used for time-lapse observation of cotyledon epidermal cell morphogenesis, but these methods require expensive microscopes and can be technically demanding. Here, we propose a silver-nano-ink coating method for time-lapse imaging and quantification of morphological changes in the epidermal cells of growing Arabidopsis thaliana cotyledons. To obtain high-resolution and wide-area cotyledon surface images, we placed the seedlings on a biaxial goniometer and adjusted the cotyledons, which were coated by dropping silver ink onto them, to be as horizontal to the focal plane as possible. The omnifocal images that had the most epidermal cell shapes in the observation area were taken at multiple points to cover the whole surface area of the cotyledon. The multi-point omnifocal images were automatically stitched, and the epidermal cells were automatically and accurately segmented by machine learning. Quantification of cell morphological features based on the segmented images demonstrated that the proposed method could quantitatively evaluate jigsaw puzzle-shaped cell growth and morphogenesis. The method was successfully applied to phenotyping of the bpp125 triple mutant, which has defective pavement cell morphogenesis. The proposed method will be useful for time-lapse non-destructive phenotyping of plant surface structures and is easier to use than the conversional methods that require fluorescent dye labeling or transformation with marker gene constructs and expensive microscopes such as the confocal laser microscope.

SUBMITTER: Kikukawa K

PROVIDER: S-EPMC8490765 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain yet poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known on how these are selected and controlled the ultimate cellular effectors of terminal differentiation. We addressed this question during late Drosophila embryogenesis when the finely tuned expression of a TF, Ovo/Shavenbaby (Svb), triggers the morphological differentiation of epidermal trichomes. We used chromatin immunoprecipitation and microarray profiling to identify Svb downstream target genes and show that Svb directly regulates a large set of terminal effectors of trichome formation. Functional assays delineated 18 Svb bound sequences driving specific expression of trichome effectors, with highly similar pattern and dynamics. Coupling computational modeling to functional dissection, we further investigated the regulatory logic of these enhancers. We find that these “terminal” enhancers harbor remarkable features with respect to their functional architectures. Trichome enhancers display weak if any clustering of Svb binding sites. Moreover, the in vivo function of each site relies on its intimate context, with a critical importance of the nucleotides flanking Svb binding sites. Finally, we identify additional cis-regulatory motifs, showing a broad diversity of positioning among trichome enhancers, and that critically contribute to their activity. Taken together, these results show that trichome formation is underpinned by unexpectedly diverse modes of regulation, and shed light on the functional architecture of enhancers governing a terminal differentiation program. Chromatin from 12-14 hour old embryos expressing svb tagged with GFP (Kondo T, Plaza S, Zanet J, Benrabah E, Valenti P, Hashimoto Y, Kobayashi S, Payre F, Kageyama Y: Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis. Science 2010, 329:336-339) was collected in duplicate. Chromatin was immunoprecipitated using an antiGFP antibody and input chromatin was used as a control.

Dataset Information

Metal-Nano-Ink Coating for Monitoring and Quantification of Cotyledon Epidermal Cell Morphogenesis.

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