17 results found
Reijne A-M, Bordeu I, Pruessner G, et al., 2019, Linear stability analysis of morphodynamics during tissue regeneration in plants, JOURNAL OF PHYSICS D-APPLIED PHYSICS, Vol: 52, ISSN: 0022-3727
Baesso P, Randall RS, Sena G, 2018, Light Sheet Fluorescence Microscopy Optimized for Long-Term Imaging of Arabidopsis Root Development., Pages: 145-163
Light sheet fluorescence microscopy (LSFM) allows sustained and repeated optical sectioning of living specimens at high spatial and temporal resolution, with minimal photodamage. Here, we describe in detail both the hardware and the software elements of a live imaging method based on LSFM and optimized for tracking and 3D scanning of Arabidopsis root tips grown vertically in physiological conditions. The system is relatively inexpensive and with minimal footprint; hence it is well suited for laboratories of any size.
Sena G, Garcia L, 2017, Specimen mounting device and method for live microscopy, WO/2017/137779
A device for mounting a living (or otherwise shape-changing or moving) specimen in a chamber for microscopic examination, the device comprising: a first part for mounting the device in or on the chamber; an inlet port for receiving a flow of a liquid medium; and a second part attached to the first part, the second part being arranged to extend into the chamber in use and to form a substantially straight channel between the second part and one or more walls of the chamber, the channel being for constraining the specimen in use; wherein a first conduit extends from the inlet port, through at least part of the second part, to an outlet hole in the second part, for conveying the liquid medium into the chamber via the outlet hole. Also provided is an assembly comprising such a device mounted in a chamber, and a method of examining a specimen using such a device.
Kral N, Li P, Oliver N, et al., 2017, Quantitative characterization of electrotropism in Arabidopsis roots, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S356-S356, ISSN: 0175-7571
Kral N, Hanna Ougolnikova A, Sena G, 2016, Externally imposed electric field enhances plant root tip regeneration, Regeneration, Vol: 3, Pages: 156-167, ISSN: 2052-4412
In plants, shoot and root regeneration can be induced in the distinctive conditions oftissue culture (in vitro), but is also observed in intact individuals (in planta) recoveringfrom tissue damage. Roots, for example, can regenerate their fully excised meristems inplanta, even in mutants with impaired apical stem cell niches. Unfortunately, to date acomprehensive understanding of regeneration in plants is still missing.Here, we provide evidence that an imposed electric field can perturb apical rootregeneration in Arabidopsis. Crucially, we explored both spatial and temporalcompetences of the stump to respond to electrical stimulation, respectively by varyingthe position of the cut and the time interval between excision and stimulation.Our data indicate that a brief pulse of an electric field parallel to the root is sufficient toincrease by up to two-fold the probability of its regeneration, and to perturb the localdistribution of the hormone auxin, as well as cell division regulation. Remarkably, theorientation of the root towards the anode or the cathode is shown to play a role.
Sena G, 2014, Stem Cells and Regeneration in Plants, NEPHRON EXPERIMENTAL NEPHROLOGY, Vol: 126, Pages: 35-39, ISSN: 1660-2129
Sena G, Frentz Z, Birnbaum KD, et al., 2011, Quantitation of cellular dynamics in growing Arabidopsis roots with light sheet microscopy., PLOS One, Vol: 6, Pages: e21303-e21303, ISSN: 1932-6203
To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics.
Sena G, Birnbaum KD, 2010, Built to rebuild: in search of organizing principles in plant regeneration., Curr Opin Genet Dev, Vol: 20, Pages: 460-465, ISSN: 0959-437X
Plants are under constant attack from insects, microbes, and other physical assaults that damage or remove body parts. Regeneration is one common strategy among plants to repair their body plan. How do organisms that are proficient at regeneration adapt their developmental programs for repatterning tissues? A new body of research employing high-resolution imaging together with cell-fate markers has led to new insights into the tissues competent to regenerate and the mechanisms that re-establish pattern. In parallel to new findings in metazoan systems, recent work in plants shows that regeneration programs commonly thought to rely on dedifferentiated cells do not need to reprogram to a ground state. Imaging studies that track the expression of regulators of the plant's proliferative centers, meristems, in conjunction with mutant analysis have shed new light on the earliest organizational cues during regenerative organ formation. One promise of plant regeneration studies is to reveal the common design attributes of programs that pattern similar organs in different developmental contexts.
Sena G, Wang X, Liu HY, et al., 2009, Organ regeneration does not require a functional stem cell niche in plants., Nature, Vol: 457, Pages: 1150-1153, ISSN: 0028-0836
Plants rely on the maintenance of stem cell niches at their apices for the continuous growth of roots and shoots. However, although the developmental plasticity of plant cells has been demonstrated, it is not known whether the stem cell niche is required for organogenesis. Here we explore the capacity of a broad range of differentiating cells to regenerate an organ without the activity of a stem cell niche. Using a root-tip regeneration system in Arabidopsis thaliana to track the molecular and functional recovery of cell fates, we show that re-specification of lost cell identities begins within hours of excision and that the function of specialized cells is restored within one day. Critically, regeneration proceeds in plants with mutations that fail to maintain the stem cell niche. These results show that stem-cell-like properties that mediate complete organ regeneration are dispersed in plant meristems and are not restricted to niches, which nonetheless seem to be necessary for indeterminate growth. This regenerative reprogramming of an entire organ without transition to a stereotypical stem cell environment has intriguing parallels to recent reports of induced transdifferentiation of specific cell types in the adult organs of animals.
Sena G, Benfey PN, 2004, A broad competence to respond to SHORT-‐ROOT as revealed by tissue-‐specific ectopic expressions, Development, Vol: 131, Pages: 2817-2826, ISSN: 0950-1991
In plants, cell fate specification depends primarily on position rather than lineage. Recent results indicate that positional information can be transmitted through intercellular trafficking of transcription factors. The SHORT ROOT (SHR) gene, a member of the GRAS family of putative transcription factors, is involved in root radial patterning in Arabidopsis. Correct radial patterning depends on the positional information transmitted through limited SHR intercellular movement and translated into cell division and specification by competent target cells. To investigate the regulation of SHR movement and the competence to respond to it, we drove expression of a translational fusion SHR::GFP using four different tissue-specific promoters. In a wild-type background, SHR::GFP was not able to move from either phloem companion cells or epidermal cells, both of which have been shown to support movement of other proteins, suggesting a requirement for tissue-specific factors for SHR movement. When expressed from its native promoter in plants with multiple endodermal layers, SHR::GFP was not able to move beyond the first endodermal layer, indicating that movement is not limited by a mechanism that recognizes boundaries between cell types. Surprisingly, movement of SHR::GFP was observed when ectopic expression from an epidermal promoter was placed in a scarecrow (scr) mutant background, revealing a possible role for SCR in limiting movement. Analysis of the competence to respond to SHR-mediated cell specification activity indicated that it was broadly distributed in the epidermal lineage, while competence to respond to the cell division activity of SHR appeared limited to the initials and involved induction of SCR. The spatial distribution of competence to respond to SHR highlights the importance of tightly regulated movement in generating the root radial pattern.
Benfey PN, Gallagher K, Paquette A, et al., 2003, Radial patterning in arabidopsis: A moving target, 62nd Annual Meeting of the Society-for-Development-Biology, Pages: 523-523, ISSN: 0012-1606
Plant embryos consist primarily of two stem-cell populations known as meristems, one that will make the root and the other that makes the shoot. Determining how the cells in these meristems are able to control their own division and the differ- entiation program of their progeny to form organs is one of the major questions of plant development. We have uncovered evi- dence for a signaling center located in the internal tissues of the Arabidopsis root that provides pattern information through cell— cell movement of a transcription factor to the surrounding cell layer. In the root of Arabidopsis, we have characterized mutations in which specific meristem cells fail to divide, or their progeny acquire the wrong identity. Analysis of mutations in the SCARE- CROW (SCR) and SHORT-ROOT (SHR) genes indicates that they are key regulators of radial patterning in the root. Both SHR and SCR are members of the GRAS family of putative transcription factors. SHR acts in a non-cell-autonomous fashion to regulate the amount of RNA that is made by the SCR gene. Analysis of SHR localization revealed protein both in the stele and in the cells immediately adjacent to it, indicating that SHR is able to move from the stele to the adjacent layer. Ectopic expression of SHR results in supernumerary cell layers and altered cell speci- fication, indicating that SHR is both necessary and sufficient for cell division and cell specification in the root meristem. Efforts to identify the mechanism of this highly regulated protein move- ment will be discussed
Sena G, Nakajima N, Jung N, et al., 2002, Analysis of Arabidopsis root pattern formation: Tissue-specific ectopic expression of the "Moving" putative transcription factor short-root, 61st Annual Meeting of the Society-for-Development-Biology, Pages: 486-487, ISSN: 0012-1606
The Arabidopsis root radial pattern is formed and maintained by well-defined asymmetric divisions of a set of stem cells (initials) ocated in the apical meristem. While it has been shown that this process is primarily dependent on positional information, little is known about the actual signaling mechanism. The short-root (shr) mutant is defective in one asymmetric division in the root meri- stem, so that the resulting radial pattern is missing one tissue (endodermis). The SHR gene, a putative transcription factor, in the root is expressed in the central tissue (stele), but not in the stem cells nor in the endodermis. We have shown (1) that the SHR protein appears to move from the stele into all the “nearest- neighbor” adjacent cells, where it is localized in the nuclei. No SHR is detectable in tissues more distant from the source stele. Nothing is known about the mechanism responsible for such nearest-neighbor movement. Moreover, it has also been shown (1) that ectopic expression of SHR can result in altered cell fates and multiplication of cell layers. Tissue-specific competence to regu- late SHR movement and to respond to SHR with alteration of cell fates and/or cell divisions seems to be part of the regulation of the positional information required for the establishment of the root radial pattern. Here we present preliminary data about ectopic expression, through tissue-specific promoters, of a fully functional protein fusion SHR-GFP. Its effect on radial pattern modification, cell fate alteration, and SHR-GFP movement will be discussed. 1. K. Nakajima et al., 2001, Nature 413, 307-311.
Nakajima K, Sena G, Nawy T, et al., 2001, Intercellular movement of the putative transcription factor SHR in root patterning, NATURE, Vol: 413, Pages: 307-311, ISSN: 0028-0836
Benfey PN, Nakajima K, Sena G, et al., 2001, Radial patterning in Arabidopsis: Signaling inside out, 60th Annual Meeting of the Society-for-Development-Biology, Pages: 288-288, ISSN: 0012-1606
In contrast to animal embryos which are miniature versions of the adult, if you look at a plant embryo it is nearly impossible to predict the form or size of the adult plant. This is because plant embryos consist primarily of two stem cell populations called meristems, one that will make the root and the other that makes the shoot. Determining how the cells in these meristems are able to control how they divide and how their progeny differentiate to form organs is one of the major questions of plant development. We have uncovered evidence for a signaling center located in the internal tissues of the Arabidopsis root that provides pattern information to the surrounding cell layers. In the root of Arabidop- sis, mutations have been found in which specific meristem cells fail to divide, or their progeny acquire the wrong identity. Analysis of mutations in the SCARECROW (SCR) and SHORT-ROOT (SHR) genes indicates that they are key regulators of radial patterning in the root. Both genes have been cloned and their expression patterns are consistent with their role in radial patterning. Analysis of tissue-specific markers indicates that SCR is primarily required for the asymmetric division that gives cortex and endodermis. The SHORT-ROOT gene is required for the asymmetric cell division responsible for formation of ground tissue as well as specification of endodermis. Both SHR and SCR are members of the GRAS family of putative transcription factors. The SHORT-ROOT gene appears to act by regulating the amount of RNA that is made by the SCARECROW gene. Surprisingly, the SHORT-ROOT gene is not expressed in the same cells as the SCARECROW gene. Instead, a novel means of cell– cell signaling appears to be responsible for the transfer of radial pattern information. Ectopic expression of SHR results in supernumerary cell layers and altered cell specification, indicating that SHR is both necessary and sufficient for cell division and cell specification in the root meristem.
Helariutta Y, Fukaki H, Wysocka-Diller J, et al., 2000, The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling, CELL, Vol: 101, Pages: 555-567, ISSN: 0092-8674
Sena G, Onado C, Cappella P, et al., 1999, Measuring the complexity of cell cycle arrest and killing of drugs: kinetics of phase-‐specific effects induced by Taxol, Cytometry, Vol: 37, Pages: 113-124, ISSN: 0196-4763
Background: Paclitaxel (Taxol) is known to act mainly in mitosis, interfering with microtubule dynamics, but effects on the other cells cycle phases have been reported also. However, a comparative picture of perturbation and killing in the G(1), S and G(2)M phases after drug treatment is lacking. The approach developed by our group tackles the problem of the complexity of cell cycle effects with the aid of a computer program simulating cell cycle progression and new quantities measuring cell-cycle arrest and death. Methods: The program generates data that were compared with those given by absolute cell counts and by different flow cytometry techniques, enabling us to follow the fate of G1 and G2M blocked cells either re-entering the cycle or dying, distinguishing cytostatic and cytotoxic effects. Apoptosis was analyzed in order to refine the description of cytotoxic effects. Results: We estimated the number of blocked and dead cells after short-term Taxol treatments in a range of concentrations and post-drug incubation times. G2M block was immediately active at low concentrations but was reversible, becoming irreversible only at the highest concentrations. G1 block became active later, allowing cell cycle progression of cells initially in G1, but was still active 48 h post-treatment, at intermediate concentrations. S-phase delay was detected after 24 h. The death rate was much higher within G1 than G2M blocked cells. Conclusions: Our analysis unraveled the complexity of cell cycle effects of the drug, and revealed the activity of G1 checkpoint, hidden by a prompter but less cytotoxic G(2)M block.
Montalenti F, Sena G, Cappella P, et al., 1998, Simulating cancer-cell kinetics after drug treatment: Application to cisplatin on ovarian carcinoma, Physical Review E, Vol: 5, ISSN: 1063-651X
The kinetics of a cancer-cell population under the effects of an antitumoral drug is a topic of particular interest. Its theoretical understanding, along with the improvement of experimental investigation techniques, can indeed play an important role in antitumoral therapies development. Starting from the analysis of flowcytometric data, with the aid of computer simulation we are able to give a detailed, quantitative description of the main kinetic parameters describing drug action on cancer cells. In this paper we describe the main features of our investigation method, showing an application to Igrov-1 ovarian carcinoma cells treated with cisplatin. Intermitotic time of phases, cell-cycle delay, and block effects with consequent repair or cell mortality, in a wide range of drug doses and recovery times, are discussed and interesting information about cisplatin action is found.
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