Changes in gene expression from postnatal (3 wk and 4 wk) to young adult (8 wk) male and female mouse liver (Mus musculus)

Microarray analysis of male and female CD-1 mouse liver was carried out at 3, 4, and 8 wk of age to elucidate developmental changes in gene expression from the pre-pubertal period to young adulthood. A large number of sex-biased and sex-independent genes showed significant changes during this developmental period. Notably, sex-independent genes involved in cell cycle, chromosome condensation, and DNA replication were down regulated from 3 wk to 8 wk, while genes associated with metal ion binding, ion transport and kinase activity were up regulated. A majority of genes showing sex differential expression in adult liver did not display sex differences prior to puberty, at which time extensive changes in sex-specific gene expression were seen, primarily in males. Thus, in male liver, 76% of male-specific genes were up regulated and 47% of female-specific genes were down regulated from 3 to 8 wk of age, whereas in female liver 67% of sex-specific genes showed no significant change in expression. In both sexes, genes up regulated from 3 to 8 wk were significantly enriched (p < E-76) in the set of genes positively regulated by the liver transcription factor HNF4α, as determined in a liver-specific HNF4α knockout mouse model, while genes down regulated during this developmental period showed significant enrichment (p < E-65) for negative regulation by HNF4α. Significant enrichment of the developmentally regulated genes in genes subject to positive and negative regulation by pituitary hormone was also observed. Nine sex-specific transcription factors showed pubertal changes in expression and may contribute to the developmental changes that onset after 3-4 wk. Overall, the observed changes in gene expression during postnatal liver development reflect the deceleration of liver growth and the induction of specialized liver functions, with widespread changes in sex-specific gene expression primarily occurring in male liver. Liver RNA isolated from the following six groups of CD-1 mice was used in the present study: 3 wk old male (M) mice (n = 10; 5 per each pool) and female (F) mice (n = 10; 5 per each pool); 4 wk old male mice (n = 12; 6 per each pool) and female mice (n = 12; 6 per each pool); 8 wk old male mice (n = 12; 6 per each pool) and female mice (n = 12; 6 per each pool). These RNA pools were used in seven separate sets of competitive hybridization experiments: 1) 3 wk M vs. 3 wk F; 2) 4 wk M vs. 4 wk F; 3) 8 wk M vs. 8 wk F; 4) 3 wk M vs. 8 wk M; 5) 4 wk M vs. 8 wk M; 6) 3 wk F vs. 8 wk F; 7) 4 wk F vs. 8 wk F. Fluorescent labeling of RNA and hybridization of the Alexa 555-labeled (green) and Alexa 647-labeled (red) aRNA samples to Agilent Mouse Gene Expression 4x44k v2 microarrays (Agilent Technology, Palo Alto, CA; catalog # G4846A-026655) were carried out, with dye swapping for each of the seven hybridization experiments to eliminate dye bias. Two microarrays, one for each mixed cDNA sample, were hybridized for each of the seven fluorescent reverse pairs, giving a total of 14 microarrays.本研究针对3、4、8周龄的雌雄CD-1小鼠肝脏开展微阵列芯片（microarray）分析，以阐明从青春期前至青年成年阶段的基因表达发育变化。大量性别偏向性基因与性别非依赖性基因在该发育阶段呈现显著表达变化。值得注意的是，参与细胞周期、染色体凝缩与DNA复制的性别非依赖性基因在3至8周龄间呈现下调表达，而与金属离子结合、离子转运及激酶活性相关的基因则呈现上调表达。多数在成年肝脏中呈现性别差异表达的基因，在青春期前并未表现出性别差异；而在青春期阶段，性别特异性基因（sex-specific genes）表达发生广泛改变，该变化主要发生在雄性个体中。因此，在雄性肝脏中，从3至8周龄阶段，76%的雄性特异性基因呈现上调表达，47%的雌性特异性基因呈现下调表达；而在雌性肝脏中，67%的性别特异性基因表达无显著变化。在雌雄个体中，3至8周龄间上调表达的基因，在肝脏特异性肝细胞核因子4α（HNF4α, Hepatocyte Nuclear Factor 4α）敲除小鼠模型中证实的、受肝脏转录因子正向调控的基因集里显著富集（p < 10^-76）；而该发育阶段下调表达的基因，则显著富集于受HNF4α负向调控的基因集（p < 10^-65）。此外，本研究还观察到发育调控基因在受垂体激素正向与负向调控的基因集里显著富集。共有9个性别特异性转录因子呈现青春期表达变化，可能参与驱动3-4周龄后出现的发育调控变化。总体而言，出生后肝脏发育过程中观测到的基因表达变化，反映了肝脏生长的减缓与特异性肝脏功能的诱导，其中性别特异性基因表达的广泛变化主要发生在雄性肝脏中。本研究使用了从以下6组CD-1小鼠中分离得到的肝脏RNA：3周龄雄性（M）小鼠（n=10；每混合样本含5只小鼠）与雌性（F）小鼠（n=10；每混合样本含5只小鼠）；4周龄雄性小鼠（n=12；每混合样本含6只小鼠）与雌性小鼠（n=12；每混合样本含6只小鼠）；8周龄雄性小鼠（n=12；每混合样本含6只小鼠）与雌性小鼠（n=12；每混合样本含6只小鼠）。这些RNA混合样本被用于7组独立的竞争性杂交实验：1）3周龄雄性 vs 3周龄雌性；2）4周龄雄性 vs 4周龄雌性；3）8周龄雄性 vs 8周龄雌性；4）3周龄雄性 vs 8周龄雄性；5）4周龄雄性 vs 8周龄雄性；6）3周龄雌性 vs 8周龄雌性；7）4周龄雌性 vs 8周龄雌性。对RNA进行荧光标记，并将Alexa 555标记（绿色）与Alexa 647标记（红色）的扩增RNA（aRNA, amplified RNA）样本与安捷伦小鼠基因表达4x44k v2微阵列芯片（Agilent Mouse Gene Expression 4x44k v2 microarrays，安捷伦科技，加利福尼亚州帕洛阿尔托；货号G4846A-026655）进行杂交；同时针对7组杂交实验均进行染料互换，以消除染料偏倚。针对7组荧光反转配对实验，每个混合互补DNA（cDNA, complementary DNA）样本均使用一张微阵列芯片进行杂交，最终共使用14张微阵列芯片。