CHAPTER 12 - Patterns of Cell Growth and Differentiation in Plants

Chapter 2 Role and Regulation of Human Tumor Suppressor SUFU in Hedgehog Signaling

Background & Aims: The Hedgehog (Hh) signaling pathway specifies patterns of cell growth and differentiation during embryogenesis, and is also involved in mature tissue homeostasis, particularly in response to injury. Recently, the seminal role of this pathway in carcinogenesis, particularly in tumors of endodermal derivation such as pancreatic cancer, has been elucidated. Currently, little is known of the distribution of Hh-responsive cells in the mature exocrine pancreas, or the requirement for this pathway in epithelial regeneration. In this study, we examined the status of the Hh signaling pathway during pancreatic regeneration following exocrine injury, and also assessed the effect of Hh pathway inhibition on pancreatic regeneration, using both pharmacologic and genetic approaches. Methods: Acute exocrine pancreatic injury was induced by caerulein injection in C57BL/6J mice. To identify Hh-responding cells, we utilized a reporter mouse harboring a lacZ "knock-in" in one of the Ptch alleles (Ptch-lacZ mice). A second group of mice was additionally treated by, a specific inhibitor of the Hh-pathway. To genetically inhibit the Hh pathway in all pancreatic epithelial cell types, we used PdxI-Cre/smoflox/flox mice, while Ela-Cre-ERT2/smoflox/flox mice were used for acinar cell-specific Hh inactivation. Pancreatic tissue was harvested on days 1-7 following injury, and processed for Xgal staining (as a surrogate for Ptch activation), as well as for quantitative real-time PCR and immunohistochemical analysis. Results: We observed striking activation of the Hh pathway during acute pancreatic injury and regeneration, confirmed by marked Shh and Ptch-lacZ expression, as well as overexpression of Hh target genes, including Ptch and Gli1. Global blockade of pancreatic Hh signaling in either cyclopamine-treated or PdxI-Cre/smoflox/flox mice led to impaired regeneration of exocrine pancreas and the abnormal persistence of a novel cell population expressing both nestin and pdx1. Acinar cell-specific inhibition of Hh signaling in Ela-Cre-ERT2/smoflox/flox mice resulted in an identical phenotype. Conclusion: Our studies confirm that the Hh pathway is activated in acute pancreatic exocrine injury and repair. Inhibition of the Hh pathway prolonged the process of exocrine pancreatic regeneration. Because chronic injury in both pancreas and other solid organs is often associated with future cancer risk, activation of the Hh pathway during pancreatic injury may play a role in the initiation of pancreatic tumorigenesis.

Plant stem cell populations, unlike their animal counterparts, do not use cell migration and oriented cell divisions to maintain their size, and therefore require a precise coordination between self-renewing divisions of stem cells, and rates of cell division and differentiation among stem cell progenitors. Shoot apical meristems (SAMs) of higher plants harbor a set of stem cells within the central zone (CZ) that divide infrequently. Stem cell daughters that are displaced towards the surrounding peripheral zone (PZ) divide at a faster rate and enter into differentiation at specific locations to form leaves or flowers. The relative ratios of cells in the CZ and the PZ are maintained, despite a constant displacement of cells from the CZ into the PZ, and subsequent allocation of cells within the PZ to form organ primordia. The mechanisms that mediate this homeostatic balance are not well understood. A homeodomain transcription factor WUSCHEL, expressed in the rib meristem (RM), located beneath the CZ, has been shown to provide nonautonomous cues for stem cell specification. By employing transient spatial manipulation and live imaging, we show that an elevated level of WUS not only induces expansion of the CZ, but also results in increased cell division rates in cells of the PZ; conversely, decreases in WUS level lead to a smaller CZ and are associated with a reduction in cell division rate. Moreover, low levels of WUS lead to enlarged organ primordia, by elevating the responsiveness of the PZ cells to the plant hormone auxin. This reveals a function of WUS in mediating the balance between differentiating and non-differentiating cells of the PZ. Regulation of stem cell numbers, growth and differentiation patterns by a single transcription factor forms a interconnected and self-correcting feedback loop to provide robustness to stem cell homeostasis in a dynamic cellular environment.

Plant stem cell populations, unlike their animal counterparts, do not use cell migration and oriented cell divisions to maintain their size, and therefore require a precise coordination between self-renewing divisions of stem cells, and rates of cell division and differentiation among stem cell progenitors. Shoot apical meristems (SAMs) of higher plants harbor a set of stem cells within the central zone (CZ) that divide infrequently. Stem cell daughters that are displaced towards the surrounding peripheral zone (PZ) divide at a faster rate and enter into differentiation at specific locations to form leaves or flowers. The relative ratios of cells in the CZ and the PZ are maintained, despite a constant displacement of cells from the CZ into the PZ, and subsequent allocation of cells within the PZ to form organ primordia. The mechanisms that mediate this homeostatic balance are not well understood. A homeodomain transcription factor WUSCHEL, expressed in the rib meristem (RM), located beneath the CZ, has been shown to provide nonautonomous cues for stem cell specification. By employing transient spatial manipulation and live imaging, we show that an elevated level of WUS not only induces expansion of the CZ, but also results in increased cell division rates in cells of the PZ; conversely, decreases in WUS level lead to a smaller CZ and are associated with a reduction in cell division rate. Moreover, low levels of WUS lead to enlarged organ primordia, by elevating the responsiveness of the PZ cells to the plant hormone auxin. This reveals a function of WUS in mediating the balance between differentiating and non-differentiating cells of the PZ. Regulation of stem cell numbers, growth and differentiation patterns by a single transcription factor forms a interconnected and self-correcting feedback loop to provide robustness to stem cell homeostasis in a dynamic cellular environment.

Differential migration and proliferation of geometrical ensembles of cell clusters

Endothelial cell differentiation in hepatocellular adenomas: implications for histopathological diagnosis

Late onset of gallbladder carcinoma with meningeal carcinomatosis

In the human body, the mechanical aspects of cell matrices are critical in maintaining cell differentiation and growth patterns. Both the static rigidity of the matrix and the stretching activity of the matrix are critical factors. Using PDMS pillars of submicrometer diameters, we first determined that the static rigidity of the matrix was measured by local contractions of 100 nm by sarcomere-like units of about 2 micrometers (Wolfenson et al., 2016).  These modular rigidity-sensing machines contained many cytoskeletal proteins like tropomyosin, a-actinin, actin and myosin. When they were missing, cells did not sense soft surfaces and grew inappropriately. When rigidity sensors were present, cells would die on soft surfaces but growth was rescued if the soft surfaces were stretched. 1-5% cyclic stretching over a frequency range of 0.01 to 10 Hz caused spreading and growth (optimum 0.1 Hz) (Cui et al., 2015). Of possible factors linked to fibroblast growth, MRTF-A (Myocardin-related transcription factor-A) moved to the nucleus in 2 hrs of cyclic stretching and reversed upon cessation; but, YAP (Yes-associated protein) moved much later. Knockdown of either MRTF-A or YAP blocked stretch-dependent growth. Thus, we suggest that the repeated pulling from a soft matrix can substitute for a stiff matrix in stimulating spreading and growth.  More generally, mechanical activation of cell substrates can be used to control cell growth and even differentiation. 

The TGF-beta superfamily of proteins regulates many different biological processes, including cell growth, differentiation and embryonic pattern formation. TGF-beta-like factors signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signalling pathway. On the inside of the cell membrane, a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different factors are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs 1, 5 and 8, whereas receptors for activin and TGF-beta target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the 'common-partner' SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Here we describe Smurf1, a new member of the Hect family of E3 ubiquitin ligases. Smurf1 selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf1 messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf1 inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf1 also enhances cellular responsiveness to the Smad2 (activin/TGF-beta) pathway. Thus, targeted ubiquitination of SMADs may serve to control both embryonic development and a wide variety of cellular responses to TGF-beta signals.

Specific interaction between Smad1 and CHIP: a surface plasmon resonance study

BackgroundTwo approaches to understanding growth during the cell cycle are single-cell studies, where growth during the cell cycle of a single cell is measured, and cell-culture studies, where growth during the cell cycle of a large number of cells as an aggregate is analyzed. Mitchison has proposed that single-cell studies, because they show variations in cell growth patterns, are more suitable for understanding cell growth during the cell cycle, and should be preferred over culture studies. Specifically, Mitchison argues that one can glean the cellular growth pattern by microscopically observing single cells during the division cycle. In contrast to Mitchison's viewpoint, it is argued here that the biological laws underlying cell growth are not to be found in single-cell studies. The cellular growth law can and should be understood by studying cells as an aggregate.ResultsThe purpose or objective of cell cycle analysis is presented and discussed. These ideas are applied to the controversy between proponents of linear growth as a possible growth pattern during the cell cycle and the proponents of exponential growth during the cell cycle. Differential (pulse) and integral (single cell) experiments are compared with regard to cell cycle analysis and it is concluded that pulse-labeling approaches are preferred over microscopic examination of cell growth for distinguishing between linear and exponential growth patterns. Even more to the point, aggregate experiments are to be preferred to single-cell studies.ConclusionThe logical consistency of exponential growth – integrating and accounting for biochemistry, cell biology, and rigorous experimental analysis – leads to the conclusion that proposals of linear growth are the result of experimental perturbations and measurement limitations. It is proposed that the universal pattern of cell growth during the cell cycle is exponential.

The developmental pathway leading to plant somatic embryogenesis (SE) is true demonstration of totipotency of plant cells. During this process, somatic cells, under appropriate conditions, divide and differentiate into embryos. This developmental pathway plays an important role as an efficient means for plant regeneration and large-scale propagation. It includes a profound reprogramming of gene expression leading to changes in cell division and differentiation patterns, becoming a suitable platform to study the morpho-physiological and molecular aspects involved in plant cell differentiation and embryo development. Plant growth regulators such as auxin, as well as stress factors and DNA methylation, are key components to induce entry into SE pathways. Proteome and transcriptome analysis allowed isolation and characterization of embryogenic-specific gene markers involved in promoting vegetative-to-embryogenic transition as well as in maturation of somatic embryos contributing to the understanding of complex relationships between inductive conditions and somatic embryo formation. This review describes current advances made, mainly at the molecular level, in discovery of the main factors involved in the induction and maturation of somatic embryos providing a basic background for understanding genetic reprogramming that is at the heart of this process. We paid special attention to extracellular protein markers during SE as well as to auxin, abscisic acid and ethylene response genes, transcriptor factors and proteins involved in embryogenic competence acquisition.

Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells

Higher plants maintain continuous development throughout their life by closely regulating the process of cell differentiation (Clark, 2001; Sablowski, 2007). In plants, the balance between undifferentiated and differentiated cell fate is managed within a stem cell niche termed the meristem. Cell differentiation in the meristem is in part controlled by genetic mechanisms. For example, mutations in CLAVATA (CLV) genes increase the number of undifferentiated cells within shoot and floral meristems leading to supernumerary organs (Clark, 2001). In contrast, mutations in genes of the homeodomain transcription factors WUSCHEL (WUS) and SHOOT-MERISTEMLESS (STM) lead to the absence of the shoot or floral meristem or its early termination through differentiation (Laux et al., 1996; Long et al., 1996). Cell differentiation in the meristem is also controlled by hormonal cues, which interfaces with gene function. For example, cytokinin treatment leads to phenotypes resembling clv mutants (Lindsay et al., 2006). Furthermore, exogenous cytokinin treatment has been shown to rescue the stm mutant phenotype and WUS protein has been shown to repress transcription of genes that act in the negative feedback pathway of cytokinin signaling (Leibfried et al., 2005; Yanai et al., 2005). The plant hormone auxin also plays a role in regulating differentiation. Auxin is thought to stimulate the initiation, development and differentiation of cells specified into organs (Teale et al., 2006). Disruption of auxin transport leads to a reduction in organ initiation and differentiation (Okada et al., 1991). In this thesis we investigate spatially regulated signaling and action of auxin and cytokinin which regulate patterning of gene expression and cell differentiation. To this end, we employed two model systems of shoot meristem initiation and development in the model plant Arabidopsis thaliana: shoot and floral meristem development and de novo shoot meristem initiation from tissue culture. Based on characterization of hormone signaling and patterning of gene expression during de novo shoot meristem initiation from tissue culture we propose a novel Turing-like model by which auxin and cytokinin interact to regulate patterning of cell differentiation. In this model, the activity of auxin, the activator of cell differentiation, is regulated by cytokinin, an inhibitor of cell differentiation. Computational models of these interactions lead to self organizing patterning of hormone response and cell differentiation as observed in experiments. In our second investigation, we show that cytokinin signaling regulates the spatial patterning of the homeodomain transcription factor WUS within the shoot meristem. We demonstrate that WUS misregulation after cytokinin treatment is mediated by both CLAVATA-dependent and independent mechanisms leading to multiple feedback loops. We reveal the presence of a cytokinin perception and signaling gradient within the shoot meristem, which spatially influences size and position of the WUS domain. Finally, we have begun to identify the molecular components required for cytokinin activation of WUS expression. Of the three characterized cytokinin receptors, only Arabidopsis Histidine Kinase 2 (AHK2) is required for WUS induction in the presence of cytokinin. In contrast, the AHK3 receptor is required for negative feedback on cytokinin signaling and thus WUS. These data reveal an unappreciated specificity in cytokinin signaling in regulating downstream targets which may be important for eliciting different cell behaviors depending on the threshold of signaling and the ratio of the three cytokinin receptors within a given cell.

Patient-derived primary cell culture and xenograft are essential tools for translational research for glioblastoma. However, characteristics of each patient derived cell line and xenograft is not extensively studied. In this study, we aim to analyze the characteristics of our glioblastoma patient-derived cell lines and xenografts based on cell surface markers and their differentiation patterns. We have established 20 glioblastoma primary cell culture lines by serum free medium containing EGF and bFGF and found that primary cell culture lines could be classified based on the expression of CD133 and CD44. Four cell lines had high expression of both CD133 and CD44. Eleven cell lines had high expression of only CD44, three cell lines had high expression of only CD133, two cell lines had low expression of both CD133 and CD44. In addition when we induce differentiation, these cell lines showed differentiation to both glial and neuronal differentiation, but differentiation patterns were different depending on each cell line. Four cell lines showed predominant neuronal differentiation and others showed predominant glial differentiation. We next investigated in vivo characteristics of glioblastoma patient derived xenografts from these established cell lines. We have injected these cell lines into NOD/Shi-scid IL2Rγ KO mouse and histopathologically analyzed characteristics of xenografts. Each xenograft well recapitulated histological features of original patients’ tumors and tumor cells remarkably invade through subventricular zone. These results suggest that glioblastoma patient derived primary cell lines and xenografts have different characteristics of cell surface marker expressions and differentiation patterns, thus can classify these cell lines depending on cell surface marker expressions and differentiation patterns. Further analysis is needed to examine the biological importance of the differences in cell surface marker expressions and differentiation patterns.