ABSTRACT
The activity of the activin/nodal signaling cascade is essential for the proper specification of germ layers during gastrulation. Many of the components of this signaling pathway have been identified, but relatively few downstream targets have been discovered. Using cDNA microarrays, we have identified a novel SH3-domain-containing gene we have named Xchef-1 that is upregulated in response to activin/nodal signaling. Xchef-1 is a direct downstream target of activin and is expressed in the marginal zones of gastrulating Xenopus embryos in a dynamic pattern reminiscent of nodal expression. At neurula stages, Xchef-1 is expressed in neural crest of the head and trunk as well as in the anterior neural plate. These domains of expression are then restricted at tailbud stages to the branchial arches, and the region of the future gall bladder.
Subject(s)
Activin Receptors/metabolism , Gene Expression Regulation, Developmental , Hemagglutinins, Viral/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Neural Crest/embryology , Transforming Growth Factor beta/metabolism , Viral Fusion Proteins/chemistry , Xenopus Proteins/biosynthesis , Xenopus Proteins/genetics , Activins/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/metabolism , Gastrula/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Models, Genetic , Molecular Sequence Data , Nodal Protein , Oligonucleotide Array Sequence Analysis , Protein Structure, Tertiary , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transcription, Genetic , Xenopus , Xenopus Proteins/chemistry , src Homology DomainsABSTRACT
In this paper, fluorescent microarray images and various analysis techniques are described to improve the microarray data acquisition processes. Signal intensities produced by rarely expressed genes are initially correctly detected, but they are often lost in corrections for background, log or ratio. Our analyses indicate that a simple correlation between the mean and median signal intensities may be the best way to eliminate inaccurate microarray signals. Unlike traditional quality control methods, the low intensity signals are retained and inaccurate signals are eliminated in this mean and median correlation. With larger amounts of microarray data being generated, it becomes increasingly more difficult to analyze data on a visual basis. Our method allows for the automatic quantitative determination of accurate and reliable signals, which can then be used for normalization. We found that a mean to median correlation of 85% or higher not only retains more data than current methods, but the retained data is more accurate than traditional thresholds or common spot flagging algorithms. We have also found that by using pin microtapping and microvibrations, we can control spot quality independent from initial PCR volume.
