SHORT-ROOT regulates vascular patterning,but not apical meristematic activity in the Arabidopsis root through cytokinin homeostasis

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.     

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue‐ or cell‐specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®‐compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue‐specific split luciferase assay for non‐invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap‐compatible split luciferase assay (ETSLA) system to investigate tissue‐specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.     

A gene trap approach to identify genes that control development

One methodology called gene trap represents a versatile strategy by which murine genes that control developmental events can be captured and identified with corresponding mutants produced at the same time. Gene trap methodology has been developed and several genes and their mutants have been analyzed, but almost all of the genes reported are those already known or murine homologs of other species. In this study, the efficiency of the gene trap methodology was improved and a novel mutant mouse strain named jumonji established which displayed an intriguing defect. Homozygous fetal mice died in utero and a significant proportion of the homozygotes showed abnormal groove formation on the neural plate and a defect in neural tube closure with a mixed genetic background of 129/Ola and BALB/c. The trapped gene believed to be responsible for these phenotypes encodes a novel nuclear protein. The results reveal that the gene trap approach can identify unknown interesting genes in murine development. The gene trap strategy, however, has several problems, the greatest of which is the difficulty in prescreening embryonic stem (ES) cells for interesting trapped genes. Recent studies are solving this problem and show that the prescreening of ES cells for genes with several characteristics is possible.     

Establishing a gene trap system mediated by T-DNA(GUS) in rice

Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless β-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hygr) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positivs transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calii induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.     

Characterization of an allelic series in the MONOPTEROS gene of arabidopsis

Patterning of numerous features of plants depends on transduction of the auxin signal. Auxin signaling is mediated by several pathways, the best understood of which relies on the function of the MONOPTEROS (MP) gene. Seven mp mutant alleles have been described in the widely used Columbia background of Arabidopsis: two extensively characterized and five only partially characterized. One of these five mp alleles appears to be extinct and thus unavailable for analysis. We show that two of the four remaining, partially characterized mp alleles reported to be in the Columbia background are in fact not in this background. We extend characterization of the remaining two Columbia alleles of mp, and we identify and characterize four new alleles of mp in the Columbia background, among which the first low‐expression allele of mp and the strongest Columbia allele of mp. These genetic resources provide the research community with new experimental opportunities for insight into the function of MP‐dependent auxin signaling in plant development. genesis 52:127–133. © 2013 Wiley Periodicals, Inc.     

Exposure to high‐concentration oxygen in the neonatal period induces abnormal retinal vascular patterning in mice

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short‐term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high‐concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration‐dependent manner. In retinas of hyperoxia‐exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short‐term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth.     

A novel triple fusion reporter system for use in gene trap mutagenesis

Gene trapping is an insertional mutagenesis strategy that allows for simultaneous gene identification and mutation in embryonic stem (ES) cells. Gene trap vectors both disrupt coding sequence and report on the genes' endogenous expression. The most popular gene trap reporter to date combines beta-galactosidase expression with neomycin resistance in a fusion protein known as beta-geo. Here we describe a refinement to this reporter that also incorporates real time fluorescent readouts. We have constructed a series of gene trap vectors incorporating a novel tripartite fusion protein consisting of EGFP, beta-galactosidase, and the neomycin or hygromycin resistance activities. Our results indicate that these triple fusions can function efficiently as reporters of endogenous trapped gene expression and subcellular localization. We show that these fusion proteins constitute versatile gene trap reporters whose activity can be detected in real time by fluorescence and in fixed tissue with a sensitive enzymatic activity.     

水稻优良性状控制基因的定位进展及其在染色体上的分布

利用分子标记可以寻找并且定位目的基因, 分子标记辅助育种技术则能够快速、高效地筛选出携带目的基因的优良品种。文章总结了近年来水稻中已被定位的优良性状控制基因及与其连锁的分子标记, 其中包括抗病虫害、抗寒、抗旱、不育、育性恢复等194个基因, 这些基因中已有14个被克隆测序。在此基础上探讨了这些基因在染色体上的分布趋势。     

The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development

We have used confocal microangiography to examine and describe the vascular anatomy of the developing zebrafish, Danio rerio. This method and the profound optical clarity of zebrafish embryos make it possible to view the entire developing vasculature with unprecedented resolution. A staged series of three-dimensional images of the vascular system were collected beginning shortly after the onset of circulation at 1 day postfertilization through early- to midlarval stages at approximately 7 days postfertilization. Blood vessels in every region of the animal were imaged at each stage, and detailed "wiring patterns" were derived describing the interconnections between every major vessel. We present an overview of these data here in this paper and in an accompanying Web site "The interactive atlas of zebrafish vascular anatomy" online at (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We find a highly dynamic but also highly stereotypic pattern of vascular connections, with different sets of primitive embryonic vessels severing connections and rewiring in new configurations according to a reproducible plan. We also find that despite variation in the details of the vascular anatomy, the basic vascular plan of the developing zebrafish shows strong similarity to that of other vertebrates. This atlas will provide an invaluable foundation for future genetic and experimental studies of vascular development in the zebrafish.     

Gene trap insertional mutagenesis in mice: New vectors and germ Line mutations in two novel genes

Insertional mutagenesis based on gene trap vectors that capture endogenous splice sites is a promising tool for functional genomics. Several groups have proposed large-scale gene trap screens, but questions remain as to the type of vectors and their design. We report a set of plasmid-encoded gene trap vectors and the disruption of two novel genes. Our results include a comparison of the relative gene trapping efficiencies of two different splice acceptor sequences in ES cells and an analysis of the structure of several gene trap insertions.     

植物功能性靶向基因标记的研究进展

随着功能基因组学的发展,分子标记技术正朝着功能性靶向基因标记的方向发展。因功能标记是根据与表型紧密相关的功能基因内部特定区域多态性基序开发而来的,所以此类标记不需要进一步的验证就可在不同的遗传背景下确定等位基因的有无。从靶向基因标记和功能标记、保守DNA和基因家族标记、转座子标记、抗性基因标记、RNA标记和靶向指纹标记几方面综述了植物功能性靶向基因标记的研究进展,旨在为分子标记的开发与应用提供理论基础。     

Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells

We employed a gene trap approach to identify genes expressed in stomatal guard cells of Arabidopsis thaliana . We examined patterns of reporter gene expression in approximately 20 000 gene trap lines, and recovered five lines with exclusive or preferential expression in stomata. The screen yielded two insertions in annotated genes, encoding the CYTOCHROME P450 86A2 (CYP86A2) mono-oxygenase, and the PLEIOTROPIC DRUG RESISTANCE 3 (AtPDR3) transporter. Expression of the trapped genes in guard cells was confirmed by RT-PCR experiments in purified stomata. Examination of homozygous mutant lines revealed that abscisic acid (ABA)-induced stomatal closure was impaired in the atpdr3 mutant. In three lines, insertions occurred outside transcribed units. Expression analysis of the genes surrounding the trapping inserts identified two genes selectively expressed in guard cells, corresponding to a PP2C PROTEIN PHOSPHATASE and an unknown expressed protein gene. Statistical analyses of the chromosomal regions tagged by the gene trap insertions revealed an over-represented [A/T]AAAG motif, previously described as an essential cis -active element for gene expression in stomata. The lines described in this work identify novel genes involved in the modulation of stomatal activity, provide useful markers for the study of developmental pathways in guard cells, and are a valuable source of guard cell-specific promoters.     

An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene

The enhancer trap technique, established in Drosophila melanogaster, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. Here we show that a Minos insertion in the Ci-Musashi gene was responsible for the altered GFP expression. Ci-Musashi showed a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis.     

Molecular control of vascular development in the zebrafish

The zebrafish is emerging as a novel model for the study of embryonic vascular development. In this review we summarize the advantages of this intriguing experimental system and the advances in our understanding of the molecular control of vascular development it has allowed.     

生物技术在林业中的应用及其影响

现代生物技术正在被越来越多地应用到林业研究和生产中,并将对林业的发展产生深远的影响。本文总结了目前在林业中应用和正在发展的生物技术,探讨了与使用这些技术有关的主要问题及争论,强调了科学地评价这些技术,以及在社会可接受的范围内进行决策的重要性。最后,本文简要指出了将来生物技术研究和应用的方向,并说明了政府应该在何种程度上帮助生物技术的发展及调节它的应用。     

A gene trap knockout of the abundant sperm tail protein, outer dense fiber 2, results in preimplantation lethality

Outer dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a widespread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein-protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice.     

Pulmonary vascular development goes awry in congenital lung abnormalities

Pulmonary vascular diseases of the newborn comprise a wide range of pathological conditions with developmental abnormalities in the pulmonary vasculature. Clinically, pulmonary arterial hypertension (PH) is characterized by persistent increased resistance of the vasculature and abnormal vascular response. The classification of PH is primarily based on clinical parameters instead of morphology and distinguishes five groups of PH. Congenital lung anomalies, such as alveolar capillary dysplasia (ACD) and PH associated with congenital diaphragmatic hernia (CDH), but also bronchopulmonary dysplasia (BPD), are classified in group three. Clearly, tight and correct regulation of pulmonary vascular development is crucial for normal lung development. Human and animal model systems have increased our knowledge and make it possible to identify and characterize affected pathways and study pivotal genes. Understanding of the normal development of the pulmonary vasculature will give new insights in the origin of the spectrum of rare diseases, such as CDH, ACD, and BPD, which render a significant clinical problem in neonatal intensive care units around the world. In this review, we describe normal pulmonary vascular development, and focus on four diseases of the newborn in which abnormal pulmonary vascular development play a critical role in morbidity and mortality. In the future perspective, we indicate the lines of research that seem to be very promising for elucidating the molecular pathways involved in the origin of congenital pulmonary vascular disease. Birth Defects Research (Part C) 102:343–358, 2014. © 2014 Wiley Periodicals, Inc.     

Genome‐wide microsatellite characterisation and marker development in Chenopodium quinoa

Microsatellites, as the tracts of repetitive DNA, are an essential constituent of the plant genome that holds important evolutionary significance, and have been extensively used to develop molecular makers for genetic analysis. To understand the microsatellite dynamics of quinoa genome and its relatives, in this study we performed a genome‐wide analysis of microsatellites in five Amaranthaceae species using available genome sequences. The results demonstrated that the microsatellites of the five Amaranthaceae species were characterised by relatively high proportions of mono‐, di‐ and trinucleotide repeats with A/T rich motifs, implying conservative organisation and composition of microsatellites in this family. Furthermore, a significant negative correlation between microsatellite frequencies and GC contents (r = ?.87) were observed. In total, 533,961 (89.57%) and 542,601 (89.86%) microsatellite loci could be used to develop simple sequence repeat (SSR) molecular markers, of which 7,178 were found to be polymorphic between the two sequenced quinoa cultivars, QQ74 and Real Blanca, through in silico PCR analysis. Finally, 15 SSR markers were randomly selected to validate their polymorphism across 12 quinoa accessions by wet‐lab PCR amplification. The newly developed genome‐wide SSR markers provide a useful resource for population genetics, gene mapping and molecular breeding studies in quinoa and beyond.     

A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control

1. Download : Download high-res image (268KB)
2. Download : Download full-size image