Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray

Abstract

cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

Figure 1

Construction of 15K mouse cDNA microarray. cDNA libraries used for EST generations and the number of ESTs collected from each library are shown (Left). ESTs were clustered by their sequence similarity, and representative cDNA clones were selected from each gene cluster. Numbers and sources of cDNA clones in the 15,264 (15K) unique gene set are shown (Right).

Figure 2

Expression profiling of 15K genes between embryo and placenta at E12.5. (A) Schematic representation of experimental procedures and pseudocolored image for a typical hybridization. After removal of maternal tissues, embryo and placenta including yolk sac and amnion were dissected out and used for RNA extraction. Radiolabeled cDNAs from each sample were hybridized separately on the 15K microarray. Hybridization was repeated three times independently for each sample. Gene expression levels in placenta were represented as a gradation of red, and those in embryo were represented as a gradation of green. Genes expressed commonly in two samples appear yellow. (B) For each gene, average expression levels (in arbitrary units) were calculated from three independent hybridizations for placenta and embryo, respectively, and displayed on a scatter plot. Genes that show significantly different expression levels between embryo and placenta at the 5% significance level (P < 0.05) are displayed as green spots, and the other genes are displayed as gray spots. Solid lines indicate 2-fold expression differences between embryo and placenta; dotted lines indicate 10-fold expression differences. Results of Northern blot analyses of selected genes are shown with coordinates in the scatter plot. A total 28 clones were examined, and Northern blot for 10 clones are shown. Spots were color coded according to their expression patterns on Northern blot: placenta-specific expression (red), embryo-specific expression (blue), commonly expressed genes (yellow), and no detectable expression in both placenta and embryo (black). Genes that show statistically significant differences are represented as squares, and ones that do not are represented as circles.

Figure 3

New placenta-specific gene. (A, Top) In situ hybridization of H3018B06 gene. Transverse section of E13.5 embryo with placenta was probed with digoxigenin-labeled RNA transcribed from H3018B06 (bar = 1 mm). (Bottom Left) Magnified view of the region marked with a rectangle in the top panel (bar = 100 μm). (Bottom Right) Control in situ hybridization with a trophoblast-specific gene, 4311 (bar = 1 mm). Am, amnion; Br, brain; Fl, forelimb; Hl, hindlimb; In, intestine; Pl, placenta. (B) Nucleotide and putative amino acid sequence of H3018B06. (C) Amino acid sequence alignment between mouse H3018B06 gene and opossum PRL gene.