Dataset Information

A protocol combining multiphoton microscopy and propidium iodide for deep 3D root meristem imaging in rice: application for the screening and identification of tissue-specific enhancer trap lines.

ABSTRACT: Background:The clear visualization of 3D organization at the cellular level in plant tissues is needed to fully understand plant development processes. Imaging tools allow the visualization of the main fluorophores and in vivo growth monitoring. Confocal microscopy coupled with the use of propidium iodide (PI) counter-staining is one of the most popular tools used to characterize the structure of root meristems in A. thaliana. However, such an approach is relatively ineffective in species with more complex and thicker root systems. Results:We adapted a PI counter-staining protocol to visualize the internal 3D architecture of rice root meristems using multiphoton microscopy. This protocol is simple and compatible with the main fluorophores (CFP, GFP and mCherry). The efficiency and applicability of this protocol were demonstrated by screening a population of 57 enhancer trap lines. We successfully characterized GFP expression in all of the lines and identified 5 lines with tissue-specific expression. Conclusions:All of these resources are now available for the rice community and represent critical tools for future studies of root development.

SUBMITTER: Bureau C

PROVIDER: S-EPMC6206838 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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A protocol combining multiphoton microscopy and propidium iodide for deep 3D root meristem imaging in rice: application for the screening and identification of tissue-specific enhancer trap lines.

Bureau Charlotte C Lanau Nadège N Ingouff Mathieu M Hassan Boukhaddaoui B Meunier Anne-Cécile AC Divol Fanchon F Sevilla Rosie R Mieulet Delphine D Dievart Anne A Périn Christophe C

Plant methods 20181029

<h4>Background</h4>The clear visualization of 3D organization at the cellular level in plant tissues is needed to fully understand plant development processes. Imaging tools allow the visualization of the main fluorophores and in vivo growth monitoring. Confocal microscopy coupled with the use of propidium iodide (PI) counter-staining is one of the most popular tools used to characterize the structure of root meristems in <i>A. thaliana</i>. However, such an approach is relatively ineffective in ...[more]

PMID: 30386414

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Project description:BACKGROUND: We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology. DESCRIPTION: To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish. CONCLUSION: zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.

Dataset Information

A protocol combining multiphoton microscopy and propidium iodide for deep 3D root meristem imaging in rice: application for the screening and identification of tissue-specific enhancer trap lines.

Publications

A protocol combining multiphoton microscopy and propidium iodide for deep 3D root meristem imaging in rice: application for the screening and identification of tissue-specific enhancer trap lines.

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