Efficient array-based identification of novel cardiac genes through differentiation of mouse ESCs

Abstract

Remarkably, although cardiac disease accounts for the largest proportion of adult mortality and morbidity in the industrialized world, the genetic programs controlling early cardiogenesis are largely incompletely understood. To better understand this process, we set out to identify genes whose expression is enriched within early cardiac fated populations, obtaining the transcriptional signatures of mouse embryonic stem cells (mESCs) at defined intervals during their differentiation along a cardiac path. We compared the RNA profiles of cardiac precursors cells (CPCs) with time-matched non-CPCs and undifferentiated mESCs, using a transgenic mESC line harboring an Nkx2-5 cardiac-specific regulatory sequence driving green fluorescent protein (GFP) to facilitate selection of CPCs. We identify 176 transcripts that are significantly elevated in their abundance within CPCs compared with other assayed populations, predicting that they will likely play a role in cardiogenesis. Of note, approximately 24% (43/176) of the cardiogenic candidate transcripts have known roles in cardiac function or development. Importantly, we evaluated the biological relevance of a significant subset 31/133 (23%) of the remaining candidate genes by in situ hybridization at multiple time points during development (embryonic day, E7.5-9.5) and report that all were expressed in key cardiac structures during cardiogenesis. Furthermore 9/31, of which many were previously uncharacterized, were detected as early as the formation of the cardiac crescent. These data demonstrate the potential power of integrating genomic approaches with mESC differentiation to illuminate developmental processes, and provides a valuable resource that may be mined to further elucidate the genetic programs underlying cardiogenesis.

Figure 1. Description Of Experimental Design.

mESCs were differentiated within hanging droplets and RNA was isolated from the corresponding undifferentiated and differentiating mESC populations at specific time points (day 0, day 4, day 6 GFP⁺, and day 6 GFP⁻; Methods). Isolated RNA populations were then hybridized to Affymetrix 430 2.0 microarrays and the resulting transcription profiles analyzed as described in Methods.

Figure 2. Determination Of Data Integrity.

A. Principle components analysis (PCA) of the four experimental groups: day 0, day 4, day 6 GFP⁺, and day 6 GFP⁻. B. Distribution of observed p-values. The dotted line corresponds to the frequency of p-values if they were uniformly distributed across all bins.

Figure 3. Confirmation of Microarray Data by qRT-PCR.

A. Geometric fold change values of the selected genes (GENE names) in the contrast of Day 6 GFP⁺ versus day 0, day 4 and day 6 GFP⁻. B. qRT-PCR of the selected genes at day 0, day 4 and day 6 GFP⁺ time points. Error bars represent the normalized standard deviation.

Figure 4. Gene Ontology of Dataset Transcripts.

Pie chart representing the relative abundance of transcripts within the dataset of genes displaying significantly different levels in the contrast of Day 6 GFP⁺ versus day 0, day 4 and day 6 GFP⁻. Transcripts were classified according to Gene Ontology (GO) terms. Annotated transcripts whose ontology could not be determined based on available information were classified as having “insufficient information”. Transcripts with no annotation were also reported as “no annotation”.

Figure 5. Many Identified Candidates are Detected in the Cardiac Crescent.

Embryonic expression of transcripts was evaluated in mice at E7.5, E8.5 and E9.5. Nkx2-5 (A-C) and Isl1 (D-F) were used as anatomical controls for cardiac expression. 4930544G21Rik (G-I), AK080047 (J-L), Rbm24 (M-O), Ppp1r3d (P-R), Tmem108 (S-U), Air (V-X), Arhgap29 (Y-AA), Igf2 (BB-DD), Igfbp5 (EE-GG) were all expressed in the cardiac crescent of E7.5 embryos (arrowheads). HT, heart tube; H, heart; OFT, out flow tract; IFT, inflow tract; CA, common atria; RV, right ventricle; CNS, central nervous system; BA, branchial arches; S, somites; OV otic vesicle; SC, spinal cord; DRG, dorsal root ganglia; E7.5-9.5; embryonic days 7.5-9.5.

Figure 6. Candidate Genes are Frequently Expressed in Cardiac Structures.

AK033658 (A–B), AI465270 (C–D), Unc45b (E–F), 1110062M06Rik (G–H), Dlk1 (I–J), 8430436O14Rik (K–L), Ctla2a (M–N), AI430856 (O–P), and A730054J21Rik (Q–R) detected in cardiac structures at E8.5 and E9.5. Taok3 (S), 9130005N14Rik (T), Gab2 (U), and BQ947020 (V) detected in cardiac structures at E9.5. HT, heart tube; H, heart; OFT, out flow tract; IFT, inflow tract; CA, common atria; RV, right ventricle; CNS, central nervous system; BA, branchial arches; S, somites; OV otic vesicle; SC, spinal cord; G, gut.