XB-ART-8720Dev Biol. August 1, 2001; 236 (1): 64-75.
Microarray-based analysis of early development in Xenopus laevis.
In order to examine transcriptional regulation globally, during early vertebrate embryonic development, we have prepared Xenopus laevis cDNA microarrays. These prototype embryonic arrays contain 864 sequenced gastrula cDNA. In order to analyze and store array data, a microarray analysis pipeline was developed and integrated with sequence analysis and annotation tools. In three independent experimental settings, we demonstrate the power of these global approaches and provide optimized protocols for their application to molecular embryology. In the first set, by comparing maternal versus zygotic transcription, we document groups of genes that are temporally regulated. This analytical approach resulted in the discovery of novel temporally regulated genes. In the second, we examine changes in gene expression spatially during development by comparing dorsal and ventral mesoderm dissected from early gastrula embryos. We have discovered novel genes with spatial enrichment from these experiments. Finally, we use the prototype microarray to examine transcriptional responses from embryonic explants treated with activin. We selected genes (two of which are novel) regulated by activin for further characterization. All results obtained by the arrays were independently tested by RT-PCR or by in situ hybridization to provide a direct assessment of the accuracy and reproducibility of these approaches in the context of molecular embryology.
PubMed ID: 11456444
Article link: Dev Biol.
Grant support: EY 12370 NEI NIH HHS , HD32105 NICHD NIH HHS
Genes referenced: cirbp eif3a eif4g1 gata4 gmnn gpd1 gpd1l gypc hnrnpa2b1 hnrnpl hnrnpu hsp90ab1 loc100498440 mapk1 nop56 orc6 pttg1ip.1 rbmx ssr2 trmt5 ttc30a washc2a
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|FIG. 1. (A) Correlation plot of the ratio of the ratios plotted against the channel intensity. The correlation of duplicate experiments is calculated as described. This plot compares the expression ratios obtained for two independent array determinations. Four similar plots can be generated from the intensity values of each of the four channels. As the intensity of the signal increases, the ratio/ratio values cluster closer to 1. (B) Array targets showing increased and decreased RNA levels grouped into different categories. The 48 up-regulated and 45 down-regulated genes were categorized as described for TIGR Rat arrays (http://www.tigr.org/docs/tigr-scripts/egad_scripts/role_report.spl) based on sequence analysis and are shown in the separate pie graphs. Color coding for the different groupings is shown on the right.|
|FIG. 2. PCR analysis of dorsal vs. ventral-induced genes. Genes identified by array analysis were examined by PCR. Dorsal marginal zones and ventral marginal zones were amplified by PCR using three different numbers of cycles, separated on acrylamide gels and analyzed by Molecular Dynamics PhosphorImager. The average is shown for results obtained by both RT-PCR and by the array analysis. ND, not determined.|
|FIG. 3. Whole-mount in situ hybridization of three genes identified in the dorsal/ventral experiment. (A–C) EIFg, (D–E) an EST (da68c11.yl), and (F–H) GPD. (A) EIFg expression at gastrula stage 10 (inset i, note stronger expression in the dorsal lip, see arrow) and expression at neurula stage 18 (B, dorsal view; C, anterior view, showing dorsal/ventral difference in expression). (D) EST expression at gastrula stage 10 (section through D, ii) and at neurula stage 18 (E). Stronger expression is seen in the posterior half of the embryo and is restricted to the dorsal half of the neural tube (see iii). (F) Expression of GPD at stage 11. Transcripts are seen throughout the embryo (coronal section shown in iv). At neurula stages, GPD is restricted to the dorsal neural tube (G, lateral view; H, anterior view. Sections in v and vi show the dorsal expression in the neural tube). a, anterior; p, posterior; d, dorsal; v, ventral; lines in (D–G) denote the angle and level of sections.|
|FIG. 4. PCR analysis of genes identified by the array. Activin treated caps (1) and untreated caps (2) were amplified by PCR as described and quantitated by Molecular Dynamics Phosphor- Imager. Ratios are given for the RT-PCR as well as for the array analysis to compare the two.|
|FIG. 5. Whole-mount in situ hybridization of a previously uncharacterized gene that is down-regulated in response to activin, EST clone 1F1. (A) At stage 18, 1F1 is expressed in ciliated cells and not in the dorsal neural tube. A coronal section (B) shows expression ventrally, in the floor plate (see arrow), but not in the notochord or dorsal neural tube (i). EST 1F1 is also expressed in ciliated cells in the skin (ii). Line in (A) denotes the angle and level of section.|
|ttc30a (tetratricopeptide repeat domain 30a) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.|