A microfluidic device and computational platform for high-throughput live imaging of gene expression.

To fully describe gene expression dynamics requires the ability to quantitatively capture expression in individual cells over time. Automated systems for acquiring and analyzing real-time images are needed to obtain unbiased data across many samples and conditions. We developed a microfluidics device, the RootArray, in which 64 Arabidopsis thaliana seedlings can be grown and their roots imaged by confocal microscopy over several days without manual intervention. To achieve high throughput, we decoupled acquisition from analysis. In the acquisition phase, we obtain images at low resolution and segment to identify regions of interest. Coordinates are communicated to the microscope to record the regions of interest at high resolution. In the analysis phase, we reconstruct three-dimensional objects from stitched high-resolution images and extract quantitative measurements from a virtual medial section of the root. We tracked hundreds of roots to capture detailed expression patterns of 12 transgenic reporter lines under different conditions.

Quantification of transcription factor expression from Arabidopsis images.

MOTIVATION: Confocal microscopy has long provided qualitative information for a variety of applications in molecular biology. Recent advances have led to extensive image datasets, which can now serve as new data sources to obtain quantitative gene expression information. In contrast to microarrays, which usually provide data for many genes at one time point, these image data provide us with expression information for only one gene, but with the advantage of high spatial and/or temporal resolution, which is often lostin microarray samples. RESULTS: We have developed a prototype for the automatic analysis of Arabidopsis confocal images, which show the expression of a single transcription factor by means of GFP reporter constructs. Using techniques from image registration, we are able to address inherent problems of non-rigid transformation and partial mapping, and obtain relative expression values for 13 different tissues in Arabidopsis roots. This provides quantitative information with high spatial resolution, which accurately represents the underlying expression values within the organism. We validate our approach on a data set of 122 images depicting expression patterns of 30 transcription factors, both in terms of registration accuracy, as well as correlation with cell-sorted microarray data. Approaches like this will be useful to lay the groundwork to reconstruct regulatory networks on the level of tissues or even individual cells. AVAILABILITY: Upon request from the authors.

EST analysis in Ginkgo biloba: an assessment of conserved developmental regulators and gymnosperm specific genes.

BACKGROUND: Ginkgo biloba L. is the only surviving member of one of the oldest living seed plant groups with medicinal, spiritual and horticultural importance worldwide. As an evolutionary relic, it displays many characters found in the early, extinct seed plants and extant cycads. To establish a molecular base to understand the evolution of seeds and pollen, we created a cDNA library and EST dataset from the reproductive structures of male (microsporangiate), female (megasporangiate), and vegetative organs (leaves) of Ginkgo biloba. RESULTS: RNA from newly emerged male and female reproductive organs and immature leaves was used to create three distinct cDNA libraries from which 6,434 ESTs were generated. These 6,434 ESTs from Ginkgo biloba were clustered into 3,830 unigenes. A comparison of our Ginkgo unigene set against the fully annotated genomes of rice and Arabidopsis, and all available ESTs in Genbank revealed that 256 Ginkgo unigenes match only genes among the gymnosperms and non-seed plants--many with multiple matches to genes in non-angiosperm plants. Conversely, another group of unigenes in Gingko had highly significant homology to transcription factors in angiosperms involved in development, including MADS box genes as well as post-transcriptional regulators. Several of the conserved developmental genes found in Ginkgo had top BLAST homology to cycad genes. We also note here the presence of ESTs in G. biloba similar to genes that to date have only been found in gymnosperms and an additional 22 Ginkgo genes common only to genes from cycads. CONCLUSION: Our analysis of an EST dataset from G. biloba revealed genes potentially unique to gymnosperms. Many of these genes showed homology to fully sequenced clones from our cycad EST dataset found in common only with gymnosperms. Other Ginkgo ESTs are similar to developmental regulators in higher plants. This work sets the stage for future studies on Ginkgo to better understand seed and pollen evolution, and to resolve the ambiguous phylogenetic relationship of G. biloba among the gymnosperms.

Specification of adaxial cell fate during maize leaf development.

Dorsoventral (adaxial/abaxial) polarity of the maize leaf is established in the meristem and is maintained throughout organ development to coordinate proper outgrowth and patterning of the leaf. rolled leaf1 (rld1) and leafbladeless1 (lbl1) are required for the specification of the adaxial/upper leaf surface. rld1 encodes a class III homeodomain-leucine zipper (HD-ZIPIII) protein whose adaxial expression is spatially defined by miRNA166-directed transcript cleavage on the abaxial side. The semi-dominant Rld1-Original (Rld1-O) mutation, which results from a single nucleotide substitution in the miRNA166 complementary site, leads to persistent expression of mutant transcripts on the abaxial site. This causes the adaxialization or partial reversal of leaf polarity. By contrast, recessive mutations in lbl1 cause the formation of abaxialized leaves. The lbl1 and Rld1-O mutations mutually suppress each other, indicating that these two genes act in the same genetic pathway. Adaxial and meristematic expression of rld1 is reduced in lbl1 mutants, indicating that lbl1 acts upstream of rld1 to specify adaxial fate during primordium development. However, rld1 expression in the vasculature of lbl1 is normal, suggesting that the specification of adaxial/abaxial polarity during vascular and primordia development is governed by separate but overlapping pathways. We also show that members of the maize yabby gene family are expressed on the adaxial side of incipient and developing leaf primordia. This expression pattern is unlike that observed in Arabidopsis, where YABBY expression is correlated with abaxial cell fate. The yabby expression patterns in lbl1 and Rld1-O mutants suggest that the yabby genes act downstream in the same pathway as lbl1 and rld1. Moreover, our observations suggest that maize yabby genes may direct lateral organ outgrowth rather than determine cell fate. We propose that a single genetic pathway involving lbl1, rld1 and the yabby genes integrates positional information within the SAM, and leads to adaxial/abaxial patterning and mediolateral outgrowth of the leaf.

Expressed sequence tag analysis in Cycas, the most primitive living seed plant.

BACKGROUND: Cycads are ancient seed plants (living fossils) with origins in the Paleozoic. Cycads are sometimes considered a 'missing link' as they exhibit characteristics intermediate between vascular non-seed plants and the more derived seed plants. Cycads have also been implicated as the source of 'Guam's dementia', possibly due to the production of S(+)-beta-methyl-alpha, beta-diaminopropionic acid (BMAA), which is an agonist of animal glutamate receptors. RESULTS: A total of 4,200 expressed sequence tags (ESTs) were created from Cycas rumphii and clustered into 2,458 contigs, of which 1,764 had low-stringency BLAST similarity to other plant genes. Among those cycad contigs with similarity to plant genes, 1,718 cycad 'hits' are to angiosperms, 1,310 match genes in gymnosperms and 734 match lower (non-seed) plants. Forty-six contigs were found that matched only genes in lower plants and gymnosperms. Upon obtaining the complete sequence from the clones of 37/46 contigs, 14 still matched only gymnosperms. Among those cycad contigs common to higher plants, ESTs were discovered that correspond to those involved in development and signaling in present-day flowering plants. We purified a cycad EST for a glutamate receptor (GLR)-like gene, as well as ESTs potentially involved in the synthesis of the GLR agonist BMAA. CONCLUSIONS: Analysis of cycad ESTs has uncovered conserved and potentially novel genes. Furthermore, the presence of a glutamate receptor agonist, as well as a glutamate receptor-like gene in cycads, supports the hypothesis that such neuroactive plant products are not merely herbivore deterrents but may also serve a role in plant signaling.

Cycads: evolutionary innovations and the role of plant-derived neurotoxins.

Cycads are an important relic from the past and represent the oldest living seed plants. Cycads have been instrumental in our understanding the evolution of angiosperms and gymnosperms because they have recognizable morphological characteristics intermediate between less-recently evolved plants such as ferns and more-derived (advanced) plants including the angiosperms. Cycads also produce several compounds that are carcinogenic and neurotoxic. Because of their unique placement in terrestrial plant evolution, molecular studies should help to define the origins of structures that led to the rise of seed plants and the role of neurotoxic compounds that are found in cycads.