Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse embryonic stem cells to identify novel cardiac genes through differentiation

ABSTRACT: Cardiac disease accounts for the largest proportion of adult mortality and morbidity in the industrialized world. However, progress toward improved clinical treatments is hampered by an incomplete understanding of the genetic programs controlling early cardiogenesis. 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) differentiating 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. Approximately 24% (43/176) of the transcripts enriched in the CPC population have known roles in cardiac function or development. Importantly, we evaluated the biological relevance of a subset 31/133 (23%) of the remaining candidate genes by in situ hybridization and report that all were expressed in key cardiac structures during cardiogenesis (embryonic day, E7.5 - 9.5), many of which were previously uncharacterized. These data demonstrate the power of mESC differentiation to model specific developmental processes and provide a valuable resource that may be mined to further elucidate the genetic programs underlying cardiogenesis. Experiment Overall Design: Transgenic mouse embryonic stem cell line (Nkx2-5-GFP, PMID:9834187) contains an Nkx2-5 cardiac-specific transcription factor driving expression of green fluorescent protein (GFP). Cells were differentiated through embryoid body formation (hanging droplet technique) as described in PMID:8155574. Following primary mouse embryonic fibroblasts (PMEF) feeder subtraction, mESCs were dissociated and resuspended in differentiation medium. Cells at this time point were designated as day 0. Following incubation for two days, the EBs were transferred, in suspension, to poly-HEMA coated tissue culture dishes to prevent cell attachment and grown in medium containing ascorbic acid PMID:12668514) which was replenished every 3 days thereafter. Experiment Overall Design: Cells were harvested on days 0, 4 and 6 in three biological replicates Experiment Overall Design: each (1a, 2a and 3a), washed in PBS for 30 minutes at 37ÂºC then resuspended in 0.25% trypsin for 7 minutes. Experiment Overall Design: Cells from day 6 were then sorted using the BD Biosciences FACSAriaâ¢ Cell sorting system into GFP+ and GFP- fractions into RLT buffer (RNeasy, Qiagen). This sorting separates cells into two groups reflecting their differentiation into those fated to a non-cardiac lineage (GFP negative) and to a cardiac lineage (GFP positive).

ORGANISM(S): Mus musculus

SUBMITTER: John Gearhart

PROVIDER: E-GEOD-10970 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cardiac muscle differentiation in vivo is guided by sequential growth factor signals, including endoderm-derived diffusible factors, impinging on cardiogenic genes in the developing mesoderm. Previously, by RNA interference in AB2.2 mouse embryonic stem cells (mESCs), we identified the endodermal transcription factor Sox17 as essential for Mesp1 induction in primitive mesoderm and subsequent cardiac muscle differentiation. However, downstream effectors of Sox17 remained to be proven functionally. In this study, we used genome-wide profiling of Sox17-dependent genes in AB2.2 cells, RNA interference, chromatin immunoprecipitation, and luciferase reporter genes to dissect this pathway. Sox17 was required not only for Hhex (a second endodermal transcription factor) but also for Cer1, a growth factor inhibitor from endoderm that, like Hhex, controls mesoderm patterning in Xenopus toward a cardiac fate. Suppressing Hhex or Cer1 blocked cardiac myogenesis, although at a later stage than induction of Mesp1/2. Hhex was required but not sufficient for Cer1 expression. Over-expression of Sox17 induced endogenous Cer1 and sequence-specific transcription of a Cer1 reporter gene. Forced expression of Cer1 was sufficient to rescue cardiac differentiation in Hhex-deficient cells. Thus, Hhex and Cer1 are indispensable components of the Sox17 pathway for cardiopoiesis in mESCs, acting at a stage downstream from Mesp1/2. Keywords: Cardiac development, Embryonic stem cells, Endoderm, Myogenesis, RNA interference Genome-wide expression profiling of Sox17-dependent genes. Mouse embryonic stem cells expressing Sox17 or luciferase shRNA were differentiated for up to 10 days by the embryoid body method [PMID:8155574], then were analysed using Affymetrix microarrays. ESCs were transduced with lentiviral vectors coexpressing enhanced green fluorescent protein (eGFP) with shRNA against Sox17, or against firefly luciferase. Transduced cells were flow-sorted based on GFP fluorescence, grown as embryoid bodies, and transferred to tissue culture plates after 4.5 days [PMID:17360443]. Cells were harvested at days 0, 2, 4, 5, 6, 8 and 10 in two biological replicates, except where noted.

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Transcription profiling of mouse embryonic stem cells to identify novel cardiac genes through differentiation

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