Transcriptional ontogeny of the developing liver

Abstract

Background: During embryogenesis the liver is derived from endodermal cells lining the digestive tract. These endodermal progenitor cells contribute to forming the parenchyma of a number of organs including the liver and pancreas. Early in organogenesis the fetal liver is populated by hematopoietic stem cells, the source for a number of blood cells including nucleated erythrocytes. A comprehensive analysis of the transcriptional changes that occur during the early stages of development to adulthood in the liver was carried out.

Results: We characterized gene expression changes in the developing mouse liver at gestational days (GD) 11.5, 12.5, 13.5, 14.5, 16.5, and 19 and in the neonate (postnatal day (PND) 7 and 32) compared to that in the adult liver (PND67) using full-genome microarrays. The fetal liver, and to a lesser extent the neonatal liver, exhibited dramatic differences in gene expression compared to adults. Canonical pathway analysis of the fetal liver signature demonstrated increases in functions important in cell replication and DNA fidelity whereas most metabolic pathways of intermediary metabolism were under expressed. Comparison of the dataset to a number of previously published microarray datasets revealed 1) a striking similarity between the fetal liver and that of the pancreas in both mice and humans, 2) a nucleated erythrocyte signature in the fetus and 3) under expression of most xenobiotic metabolism genes throughout development, with the exception of a number of transporters associated with either hematopoietic cells or cell proliferation in hepatocytes.

Conclusions: Overall, these findings reveal the complexity of gene expression changes during liver development and maturation, and provide a foundation to predict responses to chemical and drug exposure as a function of early life-stages.

Figure 1

Transcriptional ontogeny of the developing mouse liver. A. Expression changes in fetal liver genes. Fetal liver genes were identified as detailed in the Materials and Methods. The intensity scale indicates fold-changes compared to the adult controls. Red, up-regulation; green, down-regulation; black, no change. B. RT-PCR of fetal liver gene expression in mouse livers from GD14 to PND28. *, indicates statistical significance of expression changes relative to PND28 (p ≤ 0.05); ML, maternal liver. C. In situ hybridization of Makorin 1 (Mkrn1) in the GD13.5 fetus. L, liver; Lu, lung; St, stomach; M, metanephros; SC, spinal cord. D. Global gene expression in the developing mouse liver. Genes which exhibited significant differences in expression compared to adult animals were identified as detailed in the Materials and Methods.

Figure 2

Canonical pathways altered in the developing liver. A. TightCluster groups genes into 4 temporal categories during liver development. Shown are examples of clusters that group genes into one of the 4 temporal categories. B. Genes expressed at different times during development fall into unique canonical pathways. Genes in the 4 temporal groups identified using TightCluster (Figure 2A) were analyzed using IPA. Only the top 10 significant pathways in the late group are shown. C. Global view of canonical pathways altered during development. Canonical pathways significantly altered at the indicated times during development compared to adult mice were identified using IPA. D. Increased expression of genes in canonical pathways involved in DNA maintenance and cell cycle (top) and cell fate signaling (bottom). E. Changes in canonical pathways of intermediary metabolism. Left, all significantly altered pathways of metabolism. Right, up-regulated pathways. For B-E, the scale numbers are the -log(p-value) and range from < 10^-10 to not significant (NS). Yellow, altered pathway using all genes as input; red, up-regulated pathway using all up-regulated genes as input; green, down-regulated pathway using all down-regulated genes as input; black, not significant.

Figure 3

Transcriptional similarities between the fetal liver and pancreas in the mouse. A. Principal component analysis (PCA) of fetal and neonatal liver compared to a library of ~80 mouse tissues. Left, view of all mouse tissues used in the comparison. Right, enhanced view showing the trajectory of liver maturation (arrow) from stem cells to adult livers and similarity between GD19 livers and pancreas from GD18.5 and adult animals. B. Fetal liver exhibits greater similarity to pancreas than adult liver. The biological replicates were clustered using hierarchical clustering. C. Overlap in the genes differentially expressed in the fetal liver or pancreas compared to the adult liver. Fetal liver (GD19) or adult pancreas (PND60) was compared to adult livers. D.Concordance in the direction and intensity of the fold-changes in the 9919 overlapping genes from C. E. Expression of pancreas-specific genes in the developing liver. Left, expression of all genes identified as detailed in the Materials and Methods. The position of Hamp2, examined by RT-PCR is shown. Right, pancreas-specific genes up-regulated during development. Arrowheads indicate genes examined by RT-PCR. F. Sustained expression of a subset of pancreas-specific genes in the neonate. The expression of the pancreas-specific genes was examined in the C57BL/6J male mice at the indicated times in the fetus and neonate. For D-F, the intensity scale indicates fold-changes compared to the adult controls. Red, up-regulation; green, down-regulation; black, no change. G. RT-PCR of pancreas-specific gene expression in mouse livers from GD14 to PND28.

Figure 4

Transcriptional similarities between the fetal liver and pancreas in humans. A. Fetal liver exhibits greater similarity to pancreas than adult liver in humans. The biological replicates were clustered using hierarchical clustering. B. Overlap in the genes differentially expressed in the fetal liver or pancreas compared to the adult liver. C. Concordance in the direction and intensity of the fold-changes in the 1271 human overlapping genes from B. The intensity scale indicates fold-changes compared to the adult livers. Red, up-regulation; green, down-regulation. D. Common canonical pathways in mice and humans that are altered in fetal liver and pancreas compared to adult liver. Genes were divided into those depicted in the Venn diagrams in Figure 3C and Figure 4B as 1) expressed in fetal liver only, 2) common to pancreas and fetal liver and 3) in pancreas only. These 3 groups were analyzed using IPA before and after separation into up- and down-regulated genes. The same canonical pathways were compared between mice and humans. The scale is described in Figure 2 legend.

Figure 5

Identification of a nucleated erythrocyte-specific gene signature in the developing liver. A. Expression of bone marrow-specific genes in the fetal liver. Left, bone marrow-specific genes identified in the GNF Mouse GeneAtlas V3 dataset were queried for changes in the fetal/neonatal dataset. Right, bone marrow-specific genes expressed in the fetal liver with erythrocyte-associated functions in iron transport and hemoglobin synthesis. Genes confirmed by RT-PCR are indicated. B. Alteration in the signature genes for different blood cell types in the developing liver. The percentage of genes that were altered in each of the groups out of the total number of genes was compared across all of the time points. The values were compared to the expected contribution from each of the cell types based on percentage of total number of genes (right column). The figure shows the greater than expected contribution of the nucleated erythrocyte signature genes to the overall pattern. Changes in the genes for the following cell types or categories were not observed: Differentiated cells, Lymphocytes, Monocytes, Naïve T-cells. The scale represents the percentage of genes from the indicated cell type to the overall gene expression pattern. C. Expression of nucleated erythrocyte-specific genes in the fetal liver. Nucleated erythrocyte-specific genes [16] were examined for expression changes in the fetal/neonatal dataset. Genes confirmed by RT-PCR are indicated. D. RT-PCR confirmation of nucleated erythrocyte-specific gene expression in livers from GD14 to PND28.

Figure 6

Impact of liver development on xenobiotic metabolism gene expression. A.Canonical pathways involved in xenobiotic metabolism. Scale is described in Figure 2. B. Expression of Cyp genes. C. Expression of phase II conjugating genes. D. Expression of phase III transporter genes. Right, detail of up-regulated transporter genes. Arrowheads indicate genes identified as being bone marrow-specific and thus may originate from extrahepatic cells. E. Expression of nuclear receptors during development including those that regulate xenobiotic metabolism.