Mid-zone hepatocytes trade proliferation for survival via Atf4-Chop axis in early acute liver injury
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eLife Assessment
This study addresses an important question in liver biology: how zonal hepatocytes balance survival and proliferation following injury; using spatial transcriptomics, mechanistic perturbations, and functional assays, the authors propose that a mid-zone Atf4-Chop axis to Btg2 program temporarily suppresses proliferation to promote survival during APAP-induced hepatotoxicity. The idea that distinct intrahepatic zones mount tailored stress responses is conceptually significant and has implications for regeneration and toxicology. The dataset is rich and the methodology modern, but several conclusions rely on assumptions about zonation under injury, limited injury models, and incomplete functional validation of the Atf4-Chop-Btg2 axis. With targeted revisions and additional experiments, the work has the potential to provide strong mechanistic insights into liver zonation and injury responses.
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Abstract
Hepatocytes undergo extensive proliferation to facilitate liver repair after injury, yet early adaptive changes prior to proliferation remain unclear. Here, we report that during early acetaminophen (APAP)-induced liver injury (AILI), hepatocytes exhibit transient proliferation suppression, most pronounced in mid-zone hepatocytes due to zonal APAP metabolism. Using spatial transcriptomics, immunohistochemistry, and functional studies, we identified a unique mid-zone stress-response program. Central to this adaptation is the Atf4-Chop axis, which actively suppresses proliferation via the cell cycle inhibitor Btg2, prioritizing cytoprotection over cell division. This transient arrest is a critical survival strategy: halting energy-intensive proliferation during peak injury allows mid-zone hepatocytes to redirect resources towards protection, enhancing their survival in early AILI. Thus, Atf4-Chop-mediated quiescence preserves a hepatocyte reservoir necessary for subsequent regenerative proliferation and effective repair. Our findings reveal a key adaptive trade-off in mid-zone hepatocytes where transient proliferation arrest promotes early survival to enable repair.
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eLife Assessment
This study addresses an important question in liver biology: how zonal hepatocytes balance survival and proliferation following injury; using spatial transcriptomics, mechanistic perturbations, and functional assays, the authors propose that a mid-zone Atf4-Chop axis to Btg2 program temporarily suppresses proliferation to promote survival during APAP-induced hepatotoxicity. The idea that distinct intrahepatic zones mount tailored stress responses is conceptually significant and has implications for regeneration and toxicology. The dataset is rich and the methodology modern, but several conclusions rely on assumptions about zonation under injury, limited injury models, and incomplete functional validation of the Atf4-Chop-Btg2 axis. With targeted revisions and additional experiments, the work has the potential to provide …
eLife Assessment
This study addresses an important question in liver biology: how zonal hepatocytes balance survival and proliferation following injury; using spatial transcriptomics, mechanistic perturbations, and functional assays, the authors propose that a mid-zone Atf4-Chop axis to Btg2 program temporarily suppresses proliferation to promote survival during APAP-induced hepatotoxicity. The idea that distinct intrahepatic zones mount tailored stress responses is conceptually significant and has implications for regeneration and toxicology. The dataset is rich and the methodology modern, but several conclusions rely on assumptions about zonation under injury, limited injury models, and incomplete functional validation of the Atf4-Chop-Btg2 axis. With targeted revisions and additional experiments, the work has the potential to provide strong mechanistic insights into liver zonation and injury responses.
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Reviewer #1 (Public review):
Summary:
The authors present evidence that during acetaminophen (APAP)-induced liver injury, mid-zone hepatocytes activate an integrated stress response (ISR) program via Atf4 and Chop, leading to induction of Btg2. This program suppresses proliferation in the early phase of injury, prioritizing hepatocyte survival before regeneration begins. The study uses spatial transcriptomics, immunohistochemistry, CUT&RUN, and AAV overexpression to support this model.
Strengths:
(1) Innovative use of spatial transcriptomics to capture zonal differences in hepatocyte stress responses.
(2) Identification of a mid-zone specific ISR signature and candidate downstream regulator Btg2.
(3) Functional experiments with Atf4-Chop-Btg2 modulation provide causal evidence linking ISR activation to proliferation inhibition.
(4) …
Reviewer #1 (Public review):
Summary:
The authors present evidence that during acetaminophen (APAP)-induced liver injury, mid-zone hepatocytes activate an integrated stress response (ISR) program via Atf4 and Chop, leading to induction of Btg2. This program suppresses proliferation in the early phase of injury, prioritizing hepatocyte survival before regeneration begins. The study uses spatial transcriptomics, immunohistochemistry, CUT&RUN, and AAV overexpression to support this model.
Strengths:
(1) Innovative use of spatial transcriptomics to capture zonal differences in hepatocyte stress responses.
(2) Identification of a mid-zone specific ISR signature and candidate downstream regulator Btg2.
(3) Functional experiments with Atf4-Chop-Btg2 modulation provide causal evidence linking ISR activation to proliferation inhibition.
(4) Conceptually significant model that hepatocytes actively balance survival and regeneration dynamically in a zone-specific manner.
Weaknesses:
(1) Zonation definition under injury has been shown to be sustained broadly, but is not sufficiently validated and quantified, especially considering the resolution of the 10x Visium system and the potential variation of outcomes based on how to define zones.
(2) The model is built entirely in APAP injury, which specifically targets pericentral hepatocytes. It remains unclear whether the proposed mechanism applies to other liver injuries (e.g., partial hepatectomy, CCl4).
(3) Baseline proliferation appears higher than expected in homeostasis (Figure 1B), and fold change analysis (not absolute counts) may be needed to assess zonal proliferation suppression (Figure 1D).
(4) AAV-based overexpression raises potential confounds (altered CYP activity before injury) and shows incomplete penetrance that is not quantified. (Figure 5 - Figure 6).
(5) The functional link between proliferation suppression and improved survival is inferred, but direct survival /injury readouts are limited.
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Reviewer #2 (Public review):
The manuscript reports protection of midlobular hepatocytes from APAP toxicity by activation of Atf4-CHOP (Ddit3)-mediated cell cycle arrest and stress response. The authors acknowledge that their finding is unexpected because CHOP typically induces cell death. Therefore, they functionally validate several aspects of the proposed Atf4-CHOP mechanism. Along these lines, the mitigation of APAP toxicity by AAV expression of Atf4 or Btg2, the latter identified as CHOP effector, is impressive. Whether Atf4 indeed acts through CHOP and whether midlobular hepatocytes are protected because of cell cycle arrest is less clear. These and other criticisms are described in the following.
Major points:
(1) Starting with the basics, one wonders why midlobular hepatocytes manage to mount a defensive response to APAP but …
Reviewer #2 (Public review):
The manuscript reports protection of midlobular hepatocytes from APAP toxicity by activation of Atf4-CHOP (Ddit3)-mediated cell cycle arrest and stress response. The authors acknowledge that their finding is unexpected because CHOP typically induces cell death. Therefore, they functionally validate several aspects of the proposed Atf4-CHOP mechanism. Along these lines, the mitigation of APAP toxicity by AAV expression of Atf4 or Btg2, the latter identified as CHOP effector, is impressive. Whether Atf4 indeed acts through CHOP and whether midlobular hepatocytes are protected because of cell cycle arrest is less clear. These and other criticisms are described in the following.
Major points:
(1) Starting with the basics, one wonders why midlobular hepatocytes manage to mount a defensive response to APAP but pericentral hepatocytes don't. Is this because midlobular hepatocytes express the relevant Cyps (2e1, but also 1a2 and 3a11) at lower levels, which mitigates toxicity and buys them time? This would be supported by F2A but not by F3B, at least not for the most important Cyp2e1. A moderate difference is shown for Cyp1a2 expression in F3D, but is that enough to explain the different fates? Or are additional post-transcriptional effects on these Cyps at work?
(2) The evidence presented in support of cell cycle arrest of midlobular hepatocytes is not fully convincing: there is no overt difference in S and G2/M gene scores in F2F; the marker genes used for S phase and G1 to S progression in F2G are unusual. Along these lines, one wonders if spatial transcriptomics confirmed the Ki67 immunostaining results in F1 also for specific zones, not only overall, as shown in F2E?
(3) The authors conclude in line 364 that halting of proliferation by Btg2 favors survival, which raises the question of whether Btg2 knockout causes death in midlobular hepatocytes in F6K. Data addressing this question, that is, the localization and extent of tissue necrosis and ALT levels after APAP, are missing. The efficiency of the knockout of Btg2 is also not given.
(4) Related to the previous question, the BTG2 immunostaining in F6F is not convincing when compared to F6D. One also wonders if it is necessary to apply APAP to find induction of BTG2 by AAV-Ddit3?
(5) Related to the previous question, the proposed Atf4-Ddit3 axis is challenged by the lack of midlobular induction of Atf4 in the APAP scRNA-seq data published by another group, presented in S4F and G. Further analysis of AAV-Atf4 samples generated for F5 could address whether it is really Atf4 that acts on Ddit3 in APAP toxicity.
(6) Related to the previous question, the ATF4 immunostaining in F5A doesn't look convincing, with many brown pigments appearing to be outside of the nucleus.
(7) It is not ruled out that AAV expression of Atf4 or Btg2 reduces hepatocyte sensitivity to APAP by affecting the expression of the Cyps needed for activation. In other words, does AAV-Atf4 or AAV-Btg2 change the expression of any of the Cyps relevant to APAP in the 3 weeks before APAP application (F5B)?
(8) It is laudable that the authors tried to extend their findings to humans by using snRNA-seq data from a published study (line 391), but it is unclear why they didn't analyze all 10 patients in that study but instead focused on 2 and stated that this small sample number prevented drawing definitive conclusions and could therefore only be mentioned in the discussion.
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Reviewer #3 (Public review):
Summary:
This paper by Zhu et al explores zonal gene expression changes and stress responses in the liver after APAP injury. 3-6 hours after APAP, zone 2 hepatocytes demonstrate important gene expression changes. There is an increase in stress response/cell survival genes such as Hmox1, Hspa8, Atf3, and protein degradation/autophagy genes such as Ubb, Ubc, and Sqstm1. This is hypothesized to be a "stress adaption" which happens during the initial phases of acute liver injury. Furthermore, there is a spatial redistribution of Cyp450 expression that then establishes the Mid-zone as the primary site of APAP metabolism during early AILI. This particular finding was identified previously by other groups in several single-cell papers. Ddit3 (Chop) expression also increases in zone 2. The authors focused mostly on …
Reviewer #3 (Public review):
Summary:
This paper by Zhu et al explores zonal gene expression changes and stress responses in the liver after APAP injury. 3-6 hours after APAP, zone 2 hepatocytes demonstrate important gene expression changes. There is an increase in stress response/cell survival genes such as Hmox1, Hspa8, Atf3, and protein degradation/autophagy genes such as Ubb, Ubc, and Sqstm1. This is hypothesized to be a "stress adaption" which happens during the initial phases of acute liver injury. Furthermore, there is a spatial redistribution of Cyp450 expression that then establishes the Mid-zone as the primary site of APAP metabolism during early AILI. This particular finding was identified previously by other groups in several single-cell papers. Ddit3 (Chop) expression also increases in zone 2. The authors focused mostly on the Atf4-Ddit3 axis in stress adaptation. Importantly, they probe the functionality of this axis by overexpressing either ATF4 or DDIT3 using AAV tools, and they show that these manipulations block APAP-induced injury and necrosis. This is somewhat convincing evidence that these stress response proteins are probably important during injury and regeneration.
Strengths:
Overall, I think this is a useful study, showing that the Mid-lobular zone 2 hepatocytes turn on a stress-responsive gene program that suppresses proliferation, and that this is functionally important for efficient, long-term regeneration and homeostasis. This adds to the body of literature showing the importance of zone 2 cells in hepatic regeneration, and also provides an additional mechanism that tells us how they are better at surviving chemical injuries.
Weaknesses:
The main concern is that the overexpression of ATF4 and DDIT3 is causing reduced cell death and damage by APAP. This makes it harder to understand if these genes are truly increasing survival or if they are just reducing the injury caused by APAP. It may be better to perform overexpression immediately after, or at the same time as APAP delivery. Alternatively, loss-of-function experiments using AAV-shRNAs against these targets could be useful.
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