Aneuploidy as a mechanism for stress-induced liver adaptation (original) (raw)
Proliferation by a subset of hepatocytes in Hgd+/–Fah–/– mice. To investigate the mechanism of stress-induced hepatocyte proliferation, we utilized mice with mutations in the tyrosine catabolic pathway. Mice heterozygous for Hgd and deficient for Fah were maintained with an NTBC-supplemented diet, which allows these mutants to survive and maintain normal liver function (Figure 1A and ref. 23). NTBC was removed for defined periods beginning at age 3 to 4 months (Figure 1B). After 3 to 4 weeks of selection (i.e., without NTBC-supplemented diet), body weight was reduced by approximately 20% and animals appeared sickly (Figure 1C). In contrast, Hgd–/–Fah–/– controls looked healthy and maintained constant body weight. Although the majority of liver mass was pale, which is similar to tissue from Fah–/– mice, Hgd+/–Fah–/– livers were decorated with red healthy looking hepatic nodules dispersed throughout each lobe (Figure 1D). Consistent with previous results (21), livers from these mice were enriched for discrete regions of Ki67+ hepatocytes, indicating proliferation by clonal hepatic populations (Figure 1E). Additionally, 5%–10% of these nodules were associated with loss of genetic and chromosome markers, suggesting clonal aneuploidy within the nodules (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI64026DS1). Following 2 to 3 months of selection, approximately 10% of Hgd+/–Fah–/– mice regained normal body weight (Figure 1C) and livers were repopulated 75% or more with healthy tissue (Figure 1F). Moreover, in transplantation experiments, hepatocytes derived from injury-resistant Hgd+/–Fah–/– livers underwent extensive in vivo proliferation and restored liver function in Fah–/– recipients (data not shown).
Together, the data indicate that liver repopulation in Hgd+/–Fah–/– mice proceeds in a step-wise manner in response to tyrosinemia. First, the uninjured liver contains a subset of hepatocytes, which have the capacity for functional adaptation. Such cells are randomly dispersed throughout the liver. Secondly, following chronic liver injury or stress (e.g., removal of NTBC) adapted hepatocytes maintain a functional and proliferative advantage, generating healthy nodules after several weeks. Finally, in response to continued hepatic injury, injury-resistant nodules continue to proliferate and restore the entire liver mass.
Chromosome 16 is lost by injury-resistant livers. Our group previously showed that approximately 25% of regenerating hepatic nodules in Hgd+/–Fah–/– mice acquired point mutations within the WT Hgd gene, leading to loss of function (21). Lack of functional HGD therefore blocked tyrosine catabolism at the level of homogentisic acid (Figure 1A), allowing these hepatocytes to survive and proliferate during selection. Based on the high degree of aneuploidy in the murine liver (12), we hypothesized that hepatic aneuploidy could contribute to the adaptive response by Hgd+/–Fah–/– mice. The genomic sequence encoding Hgd is located centrally in the qB3 region of chromosome 16. As Hgd heterozygotes have a single functional copy of Hgd per diploid genome, aneuploid hepatocytes lacking chromosome 16 with the functional gene product would be completely devoid of HGD activity.
To test whether chromosome 16 loss contributed to liver adaptation, we first determined the baseline degree of aneuploidy by karyotyping hepatocytes from different ages of WT mice (Figure 2A). Females and males were included in this survey, so the analysis was restricted to autosomes only. Gains of each individual autosome were detected in hepatocytes from every age group: young 20- to 21-day-old mice (0.6%, range 0%–3%), adult 4- to 5-month-old mice (3%, range 0%–7%), and senior 10- to 15-month-old mice (5%, range 2%–9%). Similarly, losses of each autosome were also detected in hepatocytes from every group: young mice (3%, range 0%–7%), adults (8%, range 3%–12%), and seniors (9%, range 5%–14%). In adults, for example, gain of chromosome 3 was seen in 3% of hepatocytes, whereas chromosome 3 loss was observed in 9% of hepatocytes. Next, we karyotyped hepatocytes from Hgd+/–Fah–/– mice that were highly repopulated (≥75%) after NTBC withdrawal and resistant to injury. Aneuploidy was detected for each autosome. Compared with WT hepatocytes, the frequency of chromosome-specific gains (12%, range 5%–20%) and losses (19%, range 5%–50%) was slightly elevated (Figure 2B), which is consistent with increased aneuploidy following extensive repopulation (15). Strikingly, chromosome 16 loss in repopulated Hgd+/–Fah–/– mice was approximately 2- to 3-fold higher than that of any other autosome (Figure 2B). To exclude the possibility that the high degree of chromosome 16 aneuploidy was simply a consequence of Fah deficiency, hepatocytes from Fah–/– mice off NTBC for 6 to 9 weeks were karyotyped (Figure 1C). Chromosome gains and losses were equivalent to frequencies observed in adult/senior WT mice, indicating that Fah deficiency alone does not promote chromosome abnormalities. Overall, nearly half of hepatocytes from repopulated Hgd+/–Fah–/– mice displayed chromosome 16 aneuploidy, which was significantly higher than the degree of chromosome 16 aneuploidy observed in WT or Fah–/– mice (Figure 1D). The strong enrichment for specific loss of chromosome 16 indicates that aneuploidy contributes to hepatic functional adaptation in chronically injured Hgd+/–Fah–/– mice.
Whole chromosome aneuploidy in hepatocytes. (A) Hepatocytes isolated from healthy WT mice were karyotyped. The percentages of hepatocytes with chromosome-specific gains (gray bars) and losses (black bars) are indicated for young mice (ages 20 to 21 days; n = 3), adult mice (ages 4 to 5 months; n = 5), and senior mice (ages 10 to 15 months; n = 6). Percentages were derived from pooled data within each group. (B) Hepatocytes from fully repopulated Hgd+/–Fah–/– mice were karyotyped (n = 4); a representative profile is shown. (C) Karyotypes were also determined for hepatocytes from Fah–/– mice that were off NTBC. Percentages were derived from pooled data (n = 4). (D) Chromosome 16 aneuploidy is indicated for all mice karyotyped. Data are shown as mean ± SEM. *P <0.005; **P = 0.03.
Genomic analysis of injury-resistant hepatocytes. A major advantage of metaphase karyotyping is elucidation of chromosome identity and copy number at the single cell level. One of the disadvantages, however, is interrogation of a relatively small sample size; typically, 20 karyotypes are determined per sample. Therefore, we performed array comparative genomic hybridization (aCGH) to analyze copy number variation from thousands of cells. This approach also enables high resolution mapping of chromosomal changes. Populations of liver cells enriched for hepatocytes were isolated from nonrepopulated and repopulated mice. Both females and males were used. Hepatic DNA was mixed with sex-mismatched reference DNA derived from WT splenocytes and subsequently hybridized to aCGH microarrays containing approximately 180,000 probes evenly dispersed throughout the genome (Supplemental Table 1). Variations in chromosome copy number are presented as a log2 ratio (Figure 3). A ratio of 0 indicates equivalent relative copy number between the hepatic and reference DNA. A ratio of –1 or 1 indicates loss or gain, respectively, within the entire population of hepatic DNA. For instance, copy number variation was not detected for autosomes in mouse 1 (WT, male); however, “loss” of the X chromosome was reported because of the sex-mismatch hybridization (Figure 3).
Copy number variation in pools of hepatocytes. Alterations in chromosome copy number were assessed by aCGH analysis using populations enriched for hepatocytes isolated from WT mice (n = 2), Hgd–/–Fah–/– mice off NTBC (n = 2) and highly repopulated Hgd+/–Fah–/– mice off NTBC (n = 4). Hybridization intensity for hepatic chromosomes is plotted as log2 ratio versus sex-mismatched diploid chromosomes (derived from splenocytes). The log2 of –1 indicates chromosome loss (e.g., loss of the X chromosome in mouse 1), whereas log2 of 1 indicates chromosome gain (e.g., gain of the X chromosome in mouse 2). Copy number changes in the Y chromosome due to gender mismatch were detected but not shown in the plots.
Copy number variation was determined using hepatocyte genomic DNA purified from WT mice, Hgd–/–Fah–/– mice (off NTBC), and injury-resistant Hgd+/–Fah–/– mice. First, analysis of WT mice revealed no changes in hepatic chromosome number among the autosomes (Figure 3; mice 1, 2). The threshold for reliably detecting whole chromosome aneuploidy by aCGH is approximately 30% mosaicism. In other words, in a heterogeneous population, whole chromosome aneuploidy is only reliably detected when present in 30% or more of the cells. This limited sensitivity explains why heterogeneous chromosome-specific aneuploidy observed by karyotyping (Figure 2A), which affects chromosomes at less than 15%, is undetectable by aCGH. Secondly, Hgd–/–Fah–/– mice were maintained without NTBC for 3 months prior to DNA isolation. Livers from these mice were refractory to tyrosinemia (Figure 1C); therefore, a dominant aneuploid karyotype was not expected. As anticipated, copy number variation within autosomes was not seen for these mice (Figure 3; mice 3, 4). This result also proves that there is no specific loss of chromosome 16 associated with this genotype. Finally, aCGH analysis of hepatocytes from Hgd+/–Fah–/– mice, which were highly repopulated by revertant hepatocytes (≥75%) and resistant to injury, revealed a single dominant autosomal alteration. Mosaicism for copy number loss involving chromosome 16 was detected in each sample (Figure 3; mice 5–8). Copy number variations were not observed in any other chromosomes.
Detailed analysis of chromosome 16 revealed 2 distinct types of aneuploidy in hepatocytes from injury-resistant Hgd+/–Fah–/– mice: terminal deletion and whole chromosome loss (Figure 4, A and B). Pools of hepatocytes contained a terminal deletion with a breakpoint in the qB2 region of chromosome 16 (Figure 4A; mice 5–8). The log2 ratios in the deleted region were –0.33 to –0.40, indicating terminal deletion in 40%–50% of the cells from these mice. Importantly, whole chromosome loss was detected in 3 of 4 samples (Figure 4A; mice 5, 6, 8). The log2 ratios for the proximal segment of chromosome 16 were between 0 and –0.25, indicating whole chromosome loss in less than 30% of hepatocytes. Failure to detect the deletion event by karyotype analysis or to differentiate whole chromosome loss and rearrangement leading to the deletion reflects a limitation of the metaphase cytogenetic approach. Karyotypes are only available for cells that reach metaphase following short-term expansion in vitro, and the chromosome makeup of nonproliferating cells is not determined. Identification of both whole chromosome loss and deletion events in heterogeneous populations of cells is a major advantage of aCGH technology.
Loss of chromosome 16 in injury-resistant livers from Hgd+/–Fah–/– mice. (A) Copy number variation from aCGH analysis is shown specifically for chromosome 16. Hybridization intensity for hepatic chromosomes is plotted as log2 ratio versus sex-mismatched diploid chromosomes derived from splenocytes. Green dots indicate log2 ratio of less than 0, and red dots indicate log2 ratio of more than 0. The extent of chromosome loss is indicated along each plot (shaded purple). (B) Illustration of mosaicism for copy number loss in a heterogeneous population of cells as detected by aCGH. Whole chromosome loss and terminal deletion events are indicated. (C) Summary showing the maximum extent of chromosome 16 loss, which includes the Hgd locus.
Hepatic aneuploidy to date has been characterized in terms of gain and loss of whole chromosomes (12, 17). Deletion of the terminal region of chromosome 16 in hepatocytes from these mice is the first deletion identified by our group. We speculate that the mutagenic effects resulting from Fah deficiency contributed to chromosome 16 breakage (24). Nevertheless, Hgd was lost in cases of whole chromosome loss and terminal deletion (Figure 4C). Thus, similar to our karyotyping experiments, hepatocytes monosomic for chromosome 16 made up a substantial fraction of livers from injury-resistant Hgd+/–Fah–/– mice.