August 1, 2001;
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.
[+] show captions
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
ttc30a (tetratricopeptide repeat domain 30a) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.