mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures

Abstract

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.

Figure 1.

Several fixatives were evaluated for RNA recovery (a), RNA integrity (b), and cDNA synthesis (c). a: Kidneys from P14 mice were placed in fixative at 4°C overnight. RNA recovery is presented as percent recovery of directly homogenized tissue (error bars, 1 std). b: Five μg of the extracted total RNA was loaded on a denatured agarose gel for integrity evaluation. c: E9.5 mouse embryos were dissected and placed in fixative at 4°C overnight. Total RNA was extracted and used for synthesis of ³²P-labeled double-stranded cDNA. The cDNA was separated on an agarose gel, blotted to nylon filter, and visualized with a phosphoimager. d: Evaluation of the morphology of zinc-fixed 10-μm cryosections. Scale bar, 20 μm. n, neural tube; a, dorsal aorta.

Figure 2.

Schematic picture of the experimental procedure and the estimated total RNA content in the sample at various stages.

Figure 3.

Evaluation of the effect of T7 amplification on microarray expression profiles. a: Schematic outline of the experiment. b–d: Evaluation of preservation of transcript abundance within samples (preservation of profiles). e and f: Expression ratios between heart and kidney for nonamplified (e) and amplified (f) RNA. g: Evaluation of preservation of transcript abundance difference between samples (preservation of ratios). The ratio values are taken from the graphs in (e) and (f), respectively. h: Evaluation of the intensity dependency of the preservation of ratios. Pearson correlation coefficients of ratio-to-ratio plots are displayed on the y axis, and the log₂ mean intensities of the corresponding genes are displayed on the x axis. The graphs in c and d are generated from single experiments. The other ratio versus intensity graphs are generated from mean signal ratios of four repeated independent experiments.

Figure 4.

Schematic chart of the proof of principle experiment. Cells are isolated with LMM/LPC from: 1) the dorsal aorta, taking both endothelial and VSMCs, or from 2) local mesenchymal cells located one cell diameter further away from the vessel lumen. The samples are T7-amplified, and finally hybridized to microarrays.

Figure 5.

Endothelial and newly induced VSMCs were laser microdissected from mouse E9.5 embryonic dorsal aorta. a and b: Staining against α-SMA demonstrates smooth muscle cell differentiation in the most ventral and dorsal cells (arrows) but not yet in lateral cells. α-SMA staining is confined to a single layer of cells, which confirms that the cells are captured at the very onset of smooth muscle cell induction. c–g: Illustration of a complete laser microdissection session showing: the specimen before laser capturing (c), LMM of the EC/VSMC dorsal aorta cells (d), LPC of the dorsal aorta cells (e), LMM of the mesenchymal cells (f), and LPC of the mesenchymal cells (g). Arrows in d, f mark the track of the laser beam, arrowheads mark erythrocytes. Scale bar, 40 μm. n, neural tube; a, dorsal aorta; c, coelom; g, gut.

Figure 6.

a: False color overlay showing part of a cDNA microarray. Spots representing aorta-overexpressed genes are red and spots representing mesenchymal genes are green. b: The results are presented in a scatter plot of aorta versus mesenchymal cells. Mean expression ratios of three independent experiments are displayed on the y axis, and combined mean signal intensities are displayed on the x axis. The diagram shows the mean values of three independent experiments. Genes that were significantly (P < 0.05) overexpressed (>1.5-fold) in the aorta pool are displayed in red.