RESUMEN

A diffuse optical tomography system with chaotic laser is proposed for the three-dimensional optical phantom. The high signal-to-noise ratio is beneficial to improve the spatial resolution of diffuse optical tomography. It is essential to drive the chaotic laser as the incident light into the optical phantom. The transmitted light emitted from phantom as the detection light and a part of the incident light as the reference light to carry out cross-correlation analysis. The high-density source-detector configuration in parallel plate structure is designed for detecting targets in the phantom. The propagation of chaotic laser in the phantom is studied theoretically and experimentally based on the diffusion equation. Image reconstruction is achieved by the cross-correlation analysis of chaotic laser and the Newton-Raphson nonlinear algorithm. The performance of the proposed system has been assessed by reconstruction localization accuracy and contrast-noise-ratio. The results show that the spatial resolution of the proposed system can reach 1.5 mm and the localization error is less than 1 mm.

RESUMEN

BACKGROUND: Long-range interactions between promoters and cis-regulatory elements, such as enhancers, play critical roles in gene regulation. However, the role of three-dimensional (3D) chromatin structure in orchestrating changes in transcriptional regulation during direct cell reprogramming is not fully understood. RESULTS: Here, we performed integrated analyses of chromosomal architecture, epigenetics, and gene expression using Hi-C, promoter Capture Hi-C (PCHi-C), ChIP-seq, and RNA-seq during trans-differentiation of Pre-B cells into macrophages with a ß-estradiol inducible C/EBPαER transgene. Within 1h of ß-estradiol induction, C/EBPα translocated from the cytoplasm to the nucleus, binding to thousands of promoters and putative regulatory elements, resulting in the downregulation of Pre-B cell-specific genes and induction of macrophage-specific genes. Hi-C results were remarkably consistent throughout trans-differentiation, revealing only a small number of TAD boundary location changes, and A/B compartment switches despite significant changes in the expression of thousands of genes. PCHi-C revealed widespread changes in promoter-anchored loops with decreased interactions in parallel with decreased gene expression, and new and increased promoter-anchored interactions in parallel with increased expression of macrophage-specific genes. CONCLUSIONS: Overall, our data demonstrate that C/EBPα-induced trans-differentiation involves few changes in genome architecture at the level of TADs and A/B compartments, in contrast with widespread reorganization of thousands of promoter-anchored loops in association with changes in gene expression and cell identity.

Asunto(s)

Reprogramación Celular , Secuencias Reguladoras de Ácidos Nucleicos , Reprogramación Celular/genética , Regiones Promotoras Genéticas , Diferenciación Celular , Estradiol

RESUMEN

Root growth relies on both cell division and cell elongation, which occur in the meristem and elongation zones, respectively. SCARECROW (SCR) and SHORT-ROOT (SHR) are GRAS family genes essential for root growth and radial patterning in the Arabidopsis root. Previous studies showed that SCR and SHR promote root growth by suppressing cytokinin response in the meristem, but there is evidence that SCR expressed beyond the meristem is also required for root growth. Here we report a previously unknown role for SCR in promoting cell elongation. Consistent with this, we found that the scr mutant accumulated a higher level of reactive oxygen species (ROS) in the elongation zone, which is probably due to decreased expression of peroxidase gene 3, which consumes hydrogen peroxide in a reaction leading to Casparian strip formation. When the oxidative stress response was blocked in the scr mutant by mutation in ABSCISIC ACID 2 (ABA2) or when the redox status was ameliorated by the upbeat 1 (upb1) mutant, the root became significantly longer, with longer cells and a larger and more mitotically active meristem. Remarkably, however, the stem cell and radial patterning defects in the double mutants still persisted. Since ROS and peroxidases are essential for endodermal differentiation, these results suggest that SCR plays a role in coordinating cell elongation, endodermal differentiation, redox homeostasis and oxidative stress response in the root. We also provide evidence that this role of SCR is independent of SHR, even though they function similarly in other aspects of root growth and development.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Raíces de Plantas/crecimiento & desarrollo , Factores de Transcripción/genética , Ácido Abscísico/metabolismo , Oxidorreductasas de Alcohol/genética , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular/genética , Regulación de la Expresión Génica de las Plantas , Homeostasis , Mutación , Oxidación-Reducción , Estrés Oxidativo/fisiología , Peroxidasa/genética , Células Vegetales , Raíces de Plantas/citología , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

The generation of reproductive incompatibility between groups requires a rare genotype with low compatibility to increase in frequency. We tested the hypothesis that sexual conflict driven by the risk of polyspermy can generate compatibility groups in gamete recognition proteins (GRPs) in the sea urchin Mesocentrotus franciscanus. We examined variation in the sperm (bindin) and egg (EBR1) GRPs, how this variation influences fertilization success and how allele frequencies shift in these GRPs over time. The EBR1 gene is a large, 4595 amino acid protein made up of 27 thrombospondin type 1 domain (TSP) and 20 C1s/C1r, uEGF and bone morphogenic protein subdomain (CUB) repeats. Two TSP and two CUB repeats each demonstrate two common non-synonymous haplotypes (alleles). Sperm bindin and one of these EBR1 repeats (TSP8) shift allele frequencies from one common to two common types over an approximate 200 year interval associated with the removal of predatory sea otters and rising sea urchin abundances; the egg receptor shifts first, followed by the sperm ligand. Laboratory crosses indicate that the historically common sperm and egg gamete recognition proteins have high compatibility as do the new common proteins, with mismatches having lower compatibility. This process of creating compatibility groups sets the stage for reproductive isolation and speciation.

Asunto(s)

Especiación Genética , Células Germinativas/fisiología , Interacciones Espermatozoide-Óvulo , Strongylocentrotus/fisiología , Animales , Fertilización , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo

RESUMEN

Sugars promote lateral root formation at low levels but become inhibitory at high C/N or C/P ratios. How sugars suppress lateral root formation is unclear, however. Here we report that WOX7, a member of the WUSCHEL related homeobox (WOX) family transcription factors, inhibits lateral root development in a sugar-dependent manner. The number of lateral root primordia increased in wox7 mutants but decreased in plants over-expressing WOX7. Plants expressing the WOX7-VP16 fusion protein produced even more lateral roots than wox7, suggesting that WOX7 acts as a transcriptional repressor in lateral root development. WOX7 is expressed at all stages of lateral root development, but it is primarily involved in lateral root initiation. Consistent with this, the wox7 mutant had a higher mitotic activity only at early stages of lateral root development. Further studies suggest that WOX7 regulates lateral root development through direct repression of cell cycle genes, particularly CYCD6;1. WOX7 expression was enhanced by sugar, reduced by auxin, but did not respond to salt and mannitol. In the wox7 mutant, the effect of sugar on lateral root formation was mitigated. These results together suggest that WOX7 plays an important role in coupling the lateral root development program and sugar status in plants.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metabolismo de los Hidratos de Carbono , Proteínas de Homeodominio/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Factores de Transcripción/genética

RESUMEN

Reactive oxygen species (ROS) are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mechanisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY (SPY), an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling. SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signaling in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed.

Asunto(s)

Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Raíces de Plantas/crecimiento & desarrollo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno , Mutación , Oxidación-Reducción , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas , Receptores de Superficie Celular , Proteínas Represoras

RESUMEN

Bundle sheath (BS) cells form a single cell layer surrounding the vascular tissue in leaves. In C3 plants, photosynthesis occurs in both the BS and mesophyll cells, but the BS cells are the major sites of photosynthesis in C4 plants, whereas the mesophyll cells are only involved in CO2 fixation. Because C4 plants are more efficient photosynthetically, introduction of the C4 mechanism into C3 plants is considered a key strategy to improve crop yield. One prerequisite for such C3-to-C4 engineering is the ability to manipulate the number and physiology of the BS cells, but the molecular basis of BS cell-fate specification remains unclear. Here we report that mutations in three GRAS family transcription factors, SHORT-ROOT (SHR), SCARECROW (SCR) and SCARECROW-LIKE 23 (SCL23), affect BS cell fate in Arabidopsis thaliana. SCR and SCL23 are expressed specifically in the BS cells and act redundantly in BS cell-fate specification, but their expression pattern and function diverge at later stages of leaf development. Using ChIP-chip experiments and sugar assays, we show that SCR is primarily involved in sugar transport whereas SCL23 functions in mineral transport. SHR is also essential for BS cell-fate specification, but it is expressed in the central vascular tissue. However, the SHR protein moves into the BS cells, where it directly regulates SCR and SCL23 expression. SHR, SCR and SCL23 homologs are present in many plant species, suggesting that this developmental pathway for BS cell-fate specification is likely to be evolutionarily conserved.

Asunto(s)

Proteínas de Arabidopsis/fisiología , Arabidopsis/citología , Diferenciación Celular/genética , Factores de Transcripción/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

SHORT-ROOT (SHR) is a key regulator of radial patterning and stem-cell renewal in the Arabidopsis root. Although SHR is expressed in the stele, its function in the vascular tissue was not recognized until recently. In shr, the protoxylem is missing due to the loss of expression of microRNA165A (miR165A) and microRNA166B (miR165B). shr is also defective in lateral root formation, but the mechanism remains unclear. To dissect the SHR developmental pathway, we recently have identified its direct targets at the genome scale by chromatin immunoprecipitation followed by microarray analysis (ChIP-chip). In further studies, we have shown that SHR regulates cytokinin homeostasis through cytokinin oxidase 3 and that this role of SHR is critical to vascular patterning in the root. In this communication we report that SHR also regulates miR165A and miR166B indirectly through its effect on cytokinin homeostasis. Although cytokinin is inhibitory to root growth, the root-apical-meristem defect in shr was not alleviated by reduction of endogenous cytokinin. These results together suggest that SHR regulates vascular patterning, but not root apical meristematic activity, through cytokinin homeostasis.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Meristema/metabolismo , Raíces de Plantas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica de las Plantas , Homeostasis/genética , Homeostasis/fisiología , MicroARNs/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Factores de Transcripción/genética

RESUMEN

Sugar is essential for all cellular activities, but at high levels it inhibits growth and development. How plants balance the tradeoffs between the need for sugars and their growth inhibitory effects is poorly understood. SHORT-ROOT (SHR) and SCARECROW (SCR) are key regulators of stem cell renewal and radial patterning in the root of Arabidopsis (Arabidopsis thaliana). Recently, we identified direct targets of SHR at the genome scale. Intriguingly, among the top-ranked list, we found a number of genes that are involved in stress responses. By chromatin immunoprecipitation-polymerase chain reaction (PCR), we showed that SHR and SCR regulate a similar but not identical set of stress response genes. Consistent with this, scr and shr were found to be hypersensitive to abscisic acid (ABA). We further showed that both mutants were hypersensitive to high levels of glucose (Glc) but responded normally to high salinity and osmoticum. The endogenous levels of sucrose, Glc, and fructose were also elevated in shr and scr. Intriguingly, although shr had sugar content and developmental defects similar to those of scr, it was much less sensitive to Glc. Chromatin immunoprecipitation-PCR and reverse transcription-PCR assays as well as transgenic studies with an ABA-INSENSITIVE2 (ABI4)-ß-glucuronidase reporter construct revealed that in root, SCR, but not SHR, repressed ABI4 and ABI5 directly and specifically in the apical meristem. When combined with abi4, scr became much more tolerant of high Glc. Finally, transgenic plants expressing ABI4 under the control of the SCR promoter manifested a short-root phenotype. These results together suggest that SCR has a SHR-independent role in mitigating the sugar response and that this role of SCR is important for root growth.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metabolismo de los Hidratos de Carbono , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Metabolismo de los Hidratos de Carbono/genética , Carbohidratos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Homeostasis/efectos de los fármacos , Manitol/farmacología , Meristema/efectos de los fármacos , Meristema/genética , Meristema/crecimiento & desarrollo , Mutación/genética , Cloruro de Sodio/farmacología , Estrés Fisiológico/genética , Factores de Transcripción/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética

RESUMEN

SHORT-ROOT (SHR) is a key regulator of root growth and development in Arabidopsis (Arabidopsis thaliana). Made in the stele, the SHR protein moves into an adjacent cell layer, where it specifies endodermal cell fate; it is also essential for apical meristem maintenance, ground tissue patterning, vascular differentiation, and lateral root formation. Much has been learned about the mechanism by which SHR controls radial patterning, but how it regulates other aspects of root morphogenesis is still unclear. To dissect the SHR developmental pathway, we have determined the genome-wide locations of SHR direct targets using a chromatin immunoprecipitation followed by microarray analysis method. K-means clustering analysis not only identified additional quiescent center-specific SHR targets but also revealed a direct role for SHR in gene regulation in the pericycle and xylem. Using cell type-specific markers, we showed that in shr, the phloem and the phloem-associated pericycle expanded, whereas the xylem and xylem-associated pericycle diminished. Interestingly, we found that cytokinin level was elevated in shr and that exogenous cytokinin conferred a shr-like vascular patterning phenotype in wild-type root. By chromatin immunoprecipitation-polymerase chain reaction and reverse transcription-polymerase chain reaction assays, we showed that SHR regulates cytokinin homeostasis by directly controlling the transcription of cytokinin oxidase 3, a cytokinin catabolism enzyme preferentially expressed in the stele. Finally, overexpression of a cytokinin oxidase in shr alleviated its vascular patterning defect. On the basis of these results, we suggest that one mechanism by which SHR controls vascular patterning is the regulation of cytokinin homeostasis.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Tipificación del Cuerpo/genética , Citocininas/metabolismo , Genoma de Planta/genética , Homeostasis , Haz Vascular de Plantas/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Tipificación del Cuerpo/efectos de los fármacos , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Citocininas/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis/efectos de los fármacos , Microscopía Confocal , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Floema/citología , Floema/efectos de los fármacos , Floema/metabolismo , Haz Vascular de Plantas/efectos de los fármacos , Haz Vascular de Plantas/genética , Factores de Transcripción/genética

RESUMEN

The ability to nondestructively image and automatically phenotype complex root systems, like those of rice (Oryza sativa), is fundamental to identifying genes underlying root system architecture (RSA). Although root systems are central to plant fitness, identifying genes responsible for RSA remains an underexplored opportunity for crop improvement. Here we describe a nondestructive imaging and analysis system for automated phenotyping and trait ranking of RSA. Using this system, we image rice roots from 12 genotypes. We automatically estimate RSA traits previously identified as important to plant function. In addition, we expand the suite of features examined for RSA to include traits that more comprehensively describe monocot RSA but that are difficult to measure with traditional methods. Using 16 automatically acquired phenotypic traits for 2,297 images from 118 individuals, we observe (1) wide variation in phenotypes among the genotypes surveyed; and (2) greater intergenotype variance of RSA features than variance within a genotype. RSA trait values are integrated into a computational pipeline that utilizes supervised learning methods to determine which traits best separate two genotypes, and then ranks the traits according to their contribution to each pairwise comparison. This trait-ranking step identifies candidate traits for subsequent quantitative trait loci analysis and demonstrates that depth and average radius are key contributors to differences in rice RSA within our set of genotypes. Our results suggest a strong genetic component underlying rice RSA. This work enables the automatic phenotyping of RSA of individuals within mapping populations, providing an integrative framework for quantitative trait loci analysis of RSA.

Asunto(s)

Oryza/genética , Fenotipo , Raíces de Plantas/genética , Sitios de Carácter Cuantitativo , Genotipo , Procesamiento de Imagen Asistido por Computador , Oryza/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo

RESUMEN

Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.

Asunto(s)

Neoplasias Encefálicas/patología , Daño del ADN , Reparación del ADN , Glioma/patología , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiación , Tolerancia a Radiación , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Apoptosis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Daño del ADN/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Glioma/genética , Glioma/metabolismo , Glicoproteínas/metabolismo , Humanos , Ratones , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Trasplante de Células Madre , Trasplante Heterólogo

RESUMEN

Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy.

Asunto(s)

Glioma/irrigación sanguínea , Glioma/patología , Neovascularización Patológica/patología , Células Madre/patología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Biopsia , Glioblastoma/irrigación sanguínea , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Trasplante Heterólogo