Transcription profiling of mouse developing liver compared both to normal adult liver and non-hepatic embryonic tissues

Understanding congenital liver disease requires elucidation of the signaling pathways and transcriptional events in the developing liver. Comprehensive assessment of gene expression between 10.5 and 16.5 dpc in the developing mouse liver and comparison with adult liver and non-hepatic embryonic tissue was validated with real-time PCR and in situ hybridization. The broad nature of the analysis provides insights into patterns of genetic control of hepatogenesis. Pathways implicated in human disease are highly regulated at the transcriptional level. Rather than activating or inhibiting a pathway or biological process by altering the expression of a single signaling molecule, transcriptional changes in large numbers of genes in a pathway or process are regulated in a coordinated manner. For example, both TGF-beta and Notch signaling is inhibited during hepatogenesis not just by decreasing transcription of multiple pathway members, but also with a complementary increase in the transcription of a pathway inhibitor. Similarly, genes related to specific biological processes exhibit strong temporal synchronization in which multiple members of the pathway have similar transcriptional regulation over time. Global coordination of signaling or functional families at the transcriptional level may be a mechanism to produce robustness of the desired outcomes. In addition, this comprehensive analysis provides a database for the further study of transcriptional events during liver development by identifying liver-specific, highly regulated genes. Experiment Overall Design: In order to provide transcriptional profile of the developing liver compared both to normal adult liver and non-hepatic embryonic tissues we performed high-density microarray analysis using Affymetrix MG 430 2.0 chips for embryonic liver samples at 10.5, 11.5, 12.5, 13.5, 14.5, and 16.5 days post conception (dpc), embryo-minus liver tissues at 10.5, 11.5, 12.5, and 14.5 dpc, and normal 10-week-old adult mouse liver. Each sample consisted of at least five embryos.解析先天性肝脏疾病的致病机制，需要阐明发育中肝脏的信号通路与转录事件。本研究针对发育中小鼠肝脏在受孕后10.5至16.5天（days post conception，简称dpc）的基因表达展开全面评估，并将其与成体肝脏及非肝胚胎组织的基因表达进行对比，该评估结果通过实时荧光定量PCR（real-time PCR）与原位杂交（in situ hybridization）得到验证。本分析覆盖范围广泛，为肝脏发生过程中的基因调控模式提供了全新见解。与人类疾病相关的信号通路在转录层面受到严格调控：其调控方式并非通过改变单个信号分子的表达来激活或抑制某一通路或生物学过程，而是使通路上大量基因的转录变化以协同方式受到调控。例如，在肝脏发生过程中，转化生长因子β（TGF-beta）与Notch信号通路均受到抑制，这种抑制不仅通过下调通路中多个成员的转录水平实现，还通过互补性地上调通路抑制剂的转录来达成。类似地，与特定生物学过程相关的基因呈现出显著的时间同步性：通路中的多个成员随时间推移表现出高度相似的转录调控模式。信号通路或功能家族在转录层面的全局协同调控，或许是保障预期结果鲁棒性的一种机制。此外，本研究通过鉴定肝脏特异性、受严格调控的基因，为肝脏发育过程中转录事件的后续研究提供了专用数据库。实验整体设计：为获取发育中小鼠肝脏相较于正常成体肝脏与非肝胚胎组织的转录谱，我们采用阿菲梅特里克斯（Affymetrix）MG 430 2.0芯片，对以下样本开展高密度微阵列分析：受孕后10.5、11.5、12.5、13.5、14.5及16.5天的胚胎肝脏样本；受孕后10.5、11.5、12.5及14.5天的去肝脏胚胎组织样本；以及10周龄正常成年小鼠肝脏样本。每个样本至少包含5枚胚胎。