Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue.

Cell size distribution is highly reproducible, whereas the size of individual cells often varies greatly within a tissue. This is obvious in a population of Arabidopsis thaliana leaf epidermal cells, which ranged from 1,000 to 10,000 µm2 in size. Endoreduplication is a specialized cell cycle in which nuclear genome size (ploidy) is doubled in the absence of cell division. Although epidermal cells require endoreduplication to enhance cellular expansion, the issue of whether this mechanism is sufficient for explaining cell size distribution remains unclear due to a lack of quantitative understanding linking the occurrence of endoreduplication with cell size diversity. Here, we addressed this question by quantitatively summarizing ploidy profile and cell size distribution using a simple theoretical framework. We first found that endoreduplication dynamics is a Poisson process through cellular maturation. This finding allowed us to construct a mathematical model to predict the time evolution of a ploidy profile with a single rate constant for endoreduplication occurrence in a given time. We reproduced experimentally measured ploidy profile in both wild-type leaf tissue and endoreduplication-related mutants with this analytical solution, further demonstrating the probabilistic property of endoreduplication. We next extended the mathematical model by incorporating the element that cell size is determined according to ploidy level to examine cell size distribution. This analysis revealed that cell size is exponentially enlarged 1.5 times every endoreduplication round. Because this theoretical simulation successfully recapitulated experimentally observed cell size distributions, we concluded that Poissonian endoreduplication dynamics and exponential size-boosting are the sources of the broad cell size distribution in epidermal tissue. More generally, this study contributes to a quantitative understanding whereby stochastic dynamics generate steady-state biological heterogeneity.

The polar auxin transport inhibitor TIBA inhibits endoreduplication in dark grown spinach hypocotyls.

We addressed the question of whether an additional round of endoreduplication in dark-grown hypocotyls is a common feature in dicotyledonous plants having endopolyploid tissues. Ploidy distributions of hypocotyl tissues derived from in vitro-grown spinach (Spinacia oleracea L. cv. Atlas) seedlings grown under different light conditions were analyzed by flow cytometry. An additional round of endoreduplication (represented by 32C cells) was found in the dark-grown hypocotyl tissues. This response was inhibited by light, the intensity of which is a crucial factor for the inhibition of endoreduplication. The higher ploidy cells in cortical tissues of the dark-grown hypocotyls had larger cell sizes, suggesting that the additional round of endoreduplication contributes to hypocotyl elongation. More importantly, a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), strongly inhibits endoreduplication, not only in spinach but also in Arabidopsis. Because other polar auxin transport inhibitors or an auxin antagonist show no or mild effects, TIBA may have a specific feature that inhibits endoreduplication.

The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth.

As animals grow, many early born structures grow by cell expansion rather than cell addition; thus growth of distinct structures must be coordinated to maintain proportionality. This phenomenon is particularly widespread in the nervous system, with dendrite arbors of many neurons expanding in concert with their substrate to sustain connectivity and maintain receptive field coverage as animals grow. After rapidly growing to establish body wall coverage, dendrites of Drosophila class IV dendrite arborization (C4da) neurons grow synchronously with their substrate, the body wall epithelium, providing a system to study how proportionality is maintained during animal growth. Here, we show that the microRNA bantam (ban) ensures coordinated growth of C4da dendrites and the epithelium through regulation of epithelial endoreplication, a modified cell cycle that entails genome amplification without cell division. In Drosophila larvae, epithelial endoreplication leads to progressive changes in dendrite-extracellular matrix (ECM) and dendrite-epithelium contacts, coupling dendrite/substrate expansion and restricting dendrite growth beyond established boundaries. Moreover, changes in epithelial expression of cell adhesion molecules, including the beta-integrin myospheroid (mys), accompany this developmental transition. Finally, endoreplication and the accompanying changes in epithelial mys expression are required to constrain late-stage dendrite growth and structural plasticity. Hence, modulating epithelium-ECM attachment probably influences substrate permissivity for dendrite growth and contributes to the dendrite-substrate coupling that ensures proportional expansion of the two cell types.

Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus.

Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells.

The functional relevance of polyploidization in the skin.

Cell proliferation and differentiation are tightly coupled through the regulation of the cell division cycle. To preserve specific functional properties in differentiated cells, distinct variants of the basic mitotic cell cycle are used in various mammalian tissues, leading to the formation of polyploid cells. In this issue of Experimental Dermatology, Gandarillas and Freije discuss the evidences for polyploidization in keratinocytes, a process whose physiological relevance is now becoming evident. A better evaluation of these unconventional cell cycles is required not only to improve our understanding of the development and structure of the epidermis but also for future therapies against skin diseases.

Cycling up the epidermis: reconciling 100 years of debate.

There is likely general consensus within the skin research community that cell cycle control is critical to epidermal homeostasis and disease. The current predominant model proposes that keratinocytes switch off DNA replication and undergo cell cycle and cell growth arrest as they initiate terminal differentiation. However, this model cannot explain key physiological features of the skin, mainly why squamous differentiation prevails over proliferation in benign hyperproliferative disorders. In recent years, we have proposed an alternative model that involves mitotic slippage and endoreplication. This new model is controversial and has encountered resistance within the field. However, looking back at history, the epidermal cell cycle has been a matter of controversy and debate for around 100 years now. The accumulated data are confusing and contradictory. Our present model can explain and reconcile both old and new paradoxical observations. Here, we explain and discuss the endoreplicative cell cycle, the evidence for and against its existence in human epidermis and the important implications for skin homeostasis and disease. We show that regardless of the strengths or weaknesses of the Endoreplication Model, the existing evidence in support of the Cell Cycle Arrest Model is very weak.

The boundary of the meristematic and elongation zones in roots: endoreduplication precedes rapid cell expansion.

Plant roots consist of a meristematic zone of mitotic cells and an elongation zone of rapidly expanding cells, in which DNA replication often occurs without cell division, a process known as endoreduplication. The duration of the cell cycle and DNA replication, as measured by 5-ethynyl-2'-deoxy-uridine (EdU) incorporation, differed between the two regions (17 h in the meristematic zone, 30 h in the elongation zone). Two distinct subnuclear patterns of EdU signals, whole and speckled, marked nuclei undergoing DNA replication at early and late S phase, respectively. The boundary region between the meristematic and elongation zones was analysed by a combination of DNA replication imaging and optical estimation of the amount of DNA in each nucleus (C-value). We found a boundary cell with 4C nuclei exhibiting the whole pattern of EdU signals. Analyses of cells in the boundary region revealed that endoreduplication precedes rapid cell elongation in roots.

Endoreplication.

Developmentally programmed polyploidy occurs by at least four different mechanisms, two of which (endoreduplication and endomitosis) involve switching from mitotic cell cycles to endocycles by the selective loss of mitotic cyclin-dependent kinase (CDK) activity and bypassing many of the processes of mitosis. Here we review the mechanisms of endoreplication, focusing on recent results from Drosophila and mice.

Endoreplication and polyploidy: insights into development and disease.

Polyploid cells have genomes that contain multiples of the typical diploid chromosome number and are found in many different organisms. Studies in a variety of animal and plant developmental systems have revealed evolutionarily conserved mechanisms that control the generation of polyploidy and have recently begun to provide clues to its physiological function. These studies demonstrate that cellular polyploidy plays important roles during normal development and also contributes to human disease, particularly cancer.

Effect of blue light on endogenous isopentenyladenine and endoreduplication during photomorphogenesis and de-etiolation of tomato (Solanum lycopersicum L.) seedlings.

Light is one of the most important factor influencing plant growth and development all through their life cycle. One of the well-known light-regulated processes is de-etiolation, i.e. the switch from skotomorphogenesis to photomorphogenesis. The hormones cytokinins (CKs) play an important role during the establishment of photomorphogenesis as exogenous CKs induced photomorphogenesis of dark-grown seedlings. Most of the studies are conducted on the plant model Arabidopsis, but no or few information are available for important crop species, such as tomato (Solanum lycopersicum L.). In our study, we analyzed for the first time the endogenous CKs content in tomato hypocotyls during skotomorphogenesis, photomorphogenesis and de-etiolation. For this purpose, two tomato genotypes were used: cv. Rutgers (wild-type; WT) and its corresponding mutant (7B-1) affected in its responses to blue light (BL). Using physiological and molecular approaches, we identified that the skotomorphogenesis is characterized by an endoreduplication-mediated cell expansion, which is inhibited upon BL exposure as seen by the accumulation of trancripts encoding CycD3, key regulators of the cell cycle. Our study showed for the first time that iP (isopentenyladenine) is the CK accumulated in the tomato hypocotyl upon BL exposure, suggesting its specific role in photomorphogenesis. This result was supported by physiological experiments and gene expression data. We propose a common model to explain the role and the relationship between CKs, namely iP, and endoreduplication during de-etiolation and photomorphogenesis.

Atypical E2Fs drive atypical cell cycles.

It is well documented that polyploid cells exist in mammalian tissues such as the placenta and liver, but their function and the mechanisms for their formation have remained elusive. Two studies now identify a role for atypical E2F transcription factors in promoting polyploidy in mammals and challenge present theories about the function of polyploidy.

Endoreduplication in cervical trophoblast cells from normal pregnancies.

OBJECTIVE: Fetal cells represented by extravillous trophoblasts (EVT) obtained from the cervix by a minimally invasive procedure are important for prenatal diagnosis in early pregnancies. Endoreduplication is a duplication of chromosomes without mitosis, leading to polyploidy that might represent increased cellular metabolic activity. In this study, we estimated the normal prevalence of polyploid trophoblasts exfoliated to the cervix between 5 and 13 weeks of gestation. METHODS: Cervical samples were obtained by cytobrush, between 5 and 13 weeks of gestation from 36 randomly selected, singleton pregnancies. FISH was done with X, Y and two 21 probes. RESULTS: We diagnosed 21 pregnancies with female and 15 pregnancies with male fetal karyotypes. A mean of 15.2 (0.02%) tetraploid cells were found in pregnancies with a female fetus and a mean of 2.0 (0.003%) tetraploid cells were found in pregnancies with a male fetus. The tetraploid cells (endoreduplicated trophoblasts) were two to three times larger than the normal cells usually seen in the cervix. CONCLUSIONS: Extravillus trophoblasts tend to form endoreduplication to the ploidy level of 4c-8c of DNA. Those cells may represent a typical phenomenon in the growing placenta. Extravillus trophoblasts from female fetuses tend to form higher rates of endoreduplication.

Mycorrhizal symbiosis stimulates endoreduplication in angiosperms.

Symbiotic and parasitic relationships can alter the degree of endoreduplication in plant cells, and a limited number of studies have documented this occurrence in root cells colonized by arbuscular mycorrhizal (AM) fungi. However, this phenomenon has not been tested in a wide range of plant species, including species that are non-endopolyploid and those that do not associate with AM fungi. We grew 37 species belonging to 16 plant families, with a range of genome sizes and a range in the degree of endopolyploidy. The endoreduplication index (EI) was compared between plants that were inoculated with Glomus irregulare and plants that were not inoculated. Of the species colonized with AM fungi, 22 of the 25 species had a significant increase in endopolyploid root nuclei over non-mycorrhizal plants, including species that do not normally exhibit endopolyploidy. Changes in the EI were strongly correlated (R(2) = 0.619) with the proportion of root length colonized by arbuscules. No change was detected in the EI for the 12 non-mycorrhizal species. This work indicates that colonization by symbiotic fungi involves a mechanism to increase nuclear DNA content in roots across many angiosperm groups and is likely linked to increased metabolism and protein production.