Plasmin activity and sterile inflammation synergize to promote lethal embryonic liver degeneration

Article activity feed

1. 

   Review Commons

   Jan 15, 2026

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Reviewer #1

   Evidence, reproducibility and clarity

   The manuscript by Wu and Griffin describes a mechanism where CHD4 and BRG1, two chromatin remodelling enzymes, have antagonistic functions to regulate extracellular matrix (ECM) plasmin activity and sterile inflammatory phenotype in the endothelial cells of the developing liver. As a follow up from a previous study, the authors investigate the phenotype of embryonic-lethal endothelial-specific CHD4-knockout, leading to liver phenotype and embryo death, and the rescue of this phenotype when subsequently BRG1 is knocked-out also in the endothelium. First, the authors show that the increase in plasmin …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Reviewer #1

   Evidence, reproducibility and clarity

   The manuscript by Wu and Griffin describes a mechanism where CHD4 and BRG1, two chromatin remodelling enzymes, have antagonistic functions to regulate extracellular matrix (ECM) plasmin activity and sterile inflammatory phenotype in the endothelial cells of the developing liver. As a follow up from a previous study, the authors investigate the phenotype of embryonic-lethal endothelial-specific CHD4-knockout, leading to liver phenotype and embryo death, and the rescue of this phenotype when subsequently BRG1 is knocked-out also in the endothelium. First, the authors show that the increase in plasmin activator uPAR (which leads to ECM degradation) in CHD4-KO embryos can be rescued by BRG1-KO, and that both CHD4 and BRG1 interact with the uPAR promoter. However, the authors demonstrate that reducing plasminogen by genetic knockout is unable to rescue the CHD4-KO embryos alone, suggesting an additional mechanism. By RNAseq analysis, the authors identify sterile inflammation as another potential contributor to the lethal phenotype of CHD4-KO embryos through increased expression of ICAM-1 in endothelial cells, also showing binding of both chromatin remodellers to ICAM-1 promoter. Finally, the authors use nonsteroidal anti-inflammatory drug carprofen, alone or in combination with plasminogen genetic knockout, and demonstrate CHD4-KO lethal embryonic phenotype rescue with the combination of plasminogen reduction and inflammation reduction, highlighting the synergistic role of both ECM degradation and sterile inflammation in this genetic KO.

   The findings of the manuscript are interesting, experiments well controlled and paper well written. While the work is of potential specialist interest to the field of liver development, there are several issues which authors should address before this paper can be published:

   Major issues:

   1. The authors still see embryonic lethality of some embryos with endothelial BRG1-KO or combined endothelial CHD4/BRG1-KO - could the authors please show or at least comment in the discussion why those animals are dying?

   We observed no dead Brg1-ECko or Brg1/Chd4-ECdko embryos by E14.5. However, at E17.5, there was an 18.8% lethality rate for Brg1-ECko mutants and a 12.5% rate for Brg1/Chd4-ECdko mutants (Fig. 1B). The reasons behind the incomplete rescue of Brg1/Chd4-ECdko embryos and the cause of death in Brg1-ECko mutants remain unknown, as we have mentioned in the revised discussion (see lines 311-316).

   1. In the qRT-PCR results Fig.2c, what is each dot?

   Each dot represents transcripts acquired from a separate embryo. We have modified the figure legend for clarification.

   1. In the same figure, I would expect that in CHD4-KO there is no CHD4 transcript, and in BRG1-KO there is no BRG1 transcript, rather than the reduction shown, which seems quite noisy (though significant) - is it this a result of normalisation? Or is indeed only a certain amount of the transcript reduced?

   The VE-Cadherin Cre mouse line utilized in this study is reported to have progressive Cre expression and activity from E8.5 to E13.5 and only to reach full penetrance across all vasculature at E14.51. The liver sinusoidal ECs (LSECs) analyzed in Fig. 2C were isolated at E12.5, before Cre activity reached its full penetrance. This is likely the primary cause of the variability in gene excision seen in this panel.

   1. In the same figure, is the statistical testing performed before or after normalisation? This can introduce errors if done after normalisation.

   Normalization was performed before statistical analysis to combine relative transcript counts from embryos harvested in multiple litters. This is now clarified in our methods (see lines 486-489).

   1. In some cases, the authors show immunofluorescence images but do not specify how many biological replicates this represents (e.g. Fig.1d, 4c-d). This should be added.

   We have updated the legends for Figs. 1E, 4C-D, and 6E-F, as suggested.

   1. I also encourage the authors to present a supplementary figure with at least one other biological replicate shown for imaging data (optional).

   We appreciated this suggestion but opted not to add additional supplemental figures, which might have been confusing to readers.

   1. The plasminogen reduction by genetic modulation results in drastic changes to the embryos' appearance - is this a whole embryo KO or endothelial-specific KO? Can authors at least comment on the differences?

   The plasminogen-deficient embryos used in this study were global knockouts; this is now clarified on line 177. The Chd4-ECko embryos with varying degrees of plasminogen deficiency that are shown in Fig. 2F were dissected at E17.5, which is ~3 days after the typical time of death for Chd4-ECko embryos. This explains why the dead and partially resorbed mutants in Fig. 2F look so different from their control (Plg-/-) littermate and from the E14.5 Chd4-ECko embryos shown in Fig. 1C.

   1. In Fig.2b, do I understand correctly only 1 sample was analysed with different areas plotted on the graph? If so, this experiment should be repeated on another set of embryos to be robust, and data plotted as a mean of each embryo (rather than areas).

   Each dot represents the mean value obtained after quantifying 4 fluorescent areas within a liver section from a single embryo. The N number indicates the number of embryos used from each genotype. We have updated the figure legend accordingly.

   1. Also in some graphs, authors specify that it was more than n>x embryos, but then - what are the dots on the graph representing? Each embryo? This should be specified (e.g. Fig.2b-c, but please check this in all the figure legends).

   Thank you for this question. We have worked to clarify the legends for all our graphs. Overall, for graphs related to embryos, each dot represents data from a single embryo. Since the sample sizes vary across genotypes, we used the smallest sample size taken from the mutant groups when listing our minimum N.

   1. "we found Plaur was the only gene that was induced in CHD4-ECko LSECs at E12.5 (Figure S3D)." - I am not sure this is correct, as gene Plau is also increased in 2/3 samples?

   Although Plau transcripts were also increased in Chd4-ECko LSECs compared to control samples, our statistical analysis showed a p-value of 0.0564, which was deemed non-significant according to our cutoff criteria of p

   1. I find the title and the running title somewhat misleading and too broad; the authors should specify more detail in the title about the content of the paper - the current statement of the title is somewhat true but shown only for one genetic model and not confirmed for all types of "lethal embryonic liver degeneration".

   We have updated the title to incorporate this suggestion. The revised title is ‘Plasmin activity and sterile inflammation synergize to promote lethal embryonic liver degeneration in endothelial chromatin remodeler mutants.’ The revised running title is ‘Plasmin and inflammation in endothelial mutant livers.’

   Minor issues:

   1. If an animal licence was used, its number should be specified in the ethics or methods section

   We have added this information to the methods (see line 383).

   1. In fig.3g it is very hard to see each of the samples, could authors try to improve this graph for clarity using colours-or split Y axis - or both?

   We have revised Fig. 3G to include a split y-axis, as suggested.

   1. "This indicates that ECs can play a pro-inflammatory role in embryonic livers and highlights the need for tight regulation to ensure normal liver growth." This sentence for me is misleading, EC are producing inflammatory signals only during the CHD4-KO according to the author's data, and authors do not show such data in normal homeostasis condition. Actually, the pro-inflammatory role here seems detrimental, and ECs should not exhibit it for correct development. The authors should rephrase this to be clearer.

   The detrimental inflammation observed when Chd4 was deleted in ECs indicates that endothelial CHD4 normally suppresses inflammation during liver development (Fig. 3F-G, and 4A-B). When endothelial CHD4 functions properly, there is no excessive cytokine activation and inflammation. We have modified the sentence to help clarify this information (see lines 295-297).

   Significance

   General assessment: The study is well controlled and well written. The findings are interesting. The limitation of the findings is only 1 combination genetic model being studied, and it is unclear if the synergistic effect of sterile inflammation and ECM degradation is broadly applicable to other models, where embryo dies because of liver failure.

   Advance: The study makes an incremental advance, following up findings from a previous study. However, it is conceptually interesting.

   Audience: The audience for this manuscript would be a liver development specialist. However, broader concepts could also be applicable to liver disease.

   Expertise: I research in the field of liver regeneration and disease.

   __Reviewer #2 __

   Evidence, reproducibility and clarity

   In essence, Wu et Al find that Chd4 mutant mice exhibit embryonic liver degeneration due to uPA-mediated plasmin hyperactivity and an ICAM-1-driven hyperinflammation and that additional mutation of BRG1 opposes this liver degeneration, possibly via ICAM-1.

   Generally, this is an excellent manuscript with a very logical sequence of experiments, although it has shortcomings such as validating their findings in an independent system, ideally human, and further establishing the translational relevance. Establishing translational relevance through mechanistic experiments that identify specific inflammatory tissue pathways, such as by blocking ICAM-1 and TNF-alpha, could also define developmental aberrations as a model for broader (patho)physiology and thereby enhance the impact on the field.

   Major

   1. The embryonic and postnatal survival data of Chd4-ECko and Brg1/Chd4-Ecdko mice should be included in Fig. 1

   We revised Fig. 1 to add representative photos and lethality rates for control and mutant embryos at E17.5 (see new Fig. 1B). All Chd4-ECko embryos we dissected at E17.5 were dead, which was consistent with our previous report2. Although *Brg1/Chd4-ECdko *embryos were largely rescued at E17.5, these mutants still die soon after birth due to lung development issues, as we previously reported3.

   1. What is the impact of Chd4-ECko and Brg1/Chd4-ECdko on the multicellular microenvironment? At a minimum, IF or spatial transcriptomics for hepatocyte and biliary markers, pericytes, and other mesenchymal cells would be recommended. Can there be a distinction made on what type of endothelial cell is affected? (sinusoidal lineage, vs. venous vs. lymphatic)

   To assess whether the multicellular microenvironment of Chd4-ECko livers was altered, we performed immunostaining for various cellular markers from E12.5 to E14.5. These markers included LYVE-1 for liver sinusoids; PROX1 and E-cadherin (ECAD) for hepatocytes; CD41 for platelets and megakaryocytes; CD45 for leukocytes; CD68 and F4/80 for macrophages; MPO for neutrophils; TER119 for erythroid cells; and a-smooth muscle actin (SMA) for pericytes and smooth muscle cells (see Fig. 4D and__ Fig. R1*__). Across all the images we examined, no obvious cell-type-specific differences were observed between control and mutant livers.

   Biliary epithelial cells, which begin to differentiate at approximately E15.54, were also assessed using cytokeratin 19 (CK19) immunostaining; however, no CK19-positive cells were detected in control livers at E14.5 (see Fig. R2*). Note that although LYVE-1 is also expressed by lymphatic endothelial cells, lymphatic vessels are not yet established in the liver at E14.52. Therefore, LYVE-1 staining is appropriate for identifying liver sinusoidal ECs at this stage of development. Our data indicate that the affected vasculature in Chd4-ECko livers is predominantly localized to the liver periphery (see Fig. 1D), which LYVE-1 staining shows to be mostly populated by sinusoidal vessels (Fig. R1B and R1F).

   *Please see uploaded Response to Reviewers PDF for Figures R1 and R2

   1. The experiments showing how endothelial Chd4 loss leads to a hyperinflammatory endothelial-and potentially hepatoblast-state are important. However, the relevance of immune cell infiltration in the hematopoietic-developing liver remains unclear. Which immune cells are presumably recruited to inflame the microenvironment then? Bone-marrow-derived? This aspect would benefit from experimental clarification, for example, using migration and/or direct co-culture versus indirect cell co-culture-ideally with or without ICAM-1 blockade-in vitro assays to determine if direct crosstalk with the CD45+ immune cell compartment explains the hyperinflammatory endothelia phenotype.

   In mice, the first hematopoietic cells emerge in the yolk sac at E7.55. Subsequently, embryonic hematopoiesis takes place in the aorta-gonad-mesonephros (AGM) region and the placenta, before immature hematopoietic cells migrate to the fetal liver. After E11.0, the fetal liver becomes the main hematopoietic organ, supporting the expansion and differentiation of hematopoietic stem and progenitor cells into all mature blood cell lineages5-8. Around E16.5, hematopoietic cells migrate to the bone marrow9, so the bone marrow is not a relevant source of infiltrating immune cells in our E12.5-14.5 Chd4-ECko mutants. We therefore examined immune cell populations, including leukocytes, macrophages, and neutrophils, in Chd4-ECko livers. No enrichment of specific immune cell types was observed in Chd4-ECko livers compared with controls at E13.5-14.5 (Fig. R1). Since immune cells develop within fetal livers at this stage, these findings suggest that they are locally activated rather than recruited to Chd4-ECko livers. Moreover, because fetal livers contain a heterogeneous mixture of immature and mature hematopoietic and immune cells, appropriate in vitro cell models to assess immune cell activation in this context are currently lacking. We have added comments to the introduction to address some of these points (see lines 66-68).

   1. Related to the previous comment: Can the authors validate their findings in an independent, ideally human, cell-based system?

   To explore this, we analyzed PLAUR and ICAM1 transcripts following CHD4 and/or BRG1 knockdown in primary human umbilical vein endothelial cells (HUVECs) for 48 hours. No antagonistic regulation of either gene was detected in HUVECs (Fig. R3*). Moreover, while Icam1 transcription was antagonistically regulated by CHD4 and BRG1 in the mouse MS1 EC line (see Fig. 5A), transcriptional regulation of Plaur by these remodelers was observed only in isolated LSECs and not in cultured MS1 cells. Together, these findings demonstrate that BRG1 and CHD4 play context-specific roles when regulating Icam1 and Plaur transcription in different EC types. Furthermore, in vitro versus in vivo EC environments may additionally influence BRG1 and CHD4 activity.

   *Please see uploaded Response to Reviewers PDF for Figure R3

   1. Identifying the specific hematopoietic/immune subset could further increase the paper's impact, as it would more definitively clarify the mechanism in the developing endothelial niche.

   Please see our response to question # 3.

   1. Also, can the authors show experimentally whether, conversely, Chd4 overexpression can limit an endothelial-type of inflammatory liver injury?

   We agree that exploring this suggestion would provide useful insights. However, we currently lack a genetic or inducible endothelial-specific Chd4 overexpression model, which makes it challenging to link our embryonic findings to the context of adult liver injury. For now, our study demonstrates that hepatic ECs regulate sterile inflammation to support embryonic liver development. Future development of appropriate genetic tools will allow us to determine if the role of endothelial CHD4 that is demonstrated in the current study is recapitulated in adult inflammatory liver injury models.

   Minor

   1. A separate figure panel for Chd4fl/fl; Vav-Cre+ appears reasonable, instead of being shown as a table.

   Thank you. Please see our new Fig. S1, which includes representative images (and lethality rates) of control and Chd4fl/fl;Vav-Cre+ embryos at E18.5.

   Significance:

   Generally, this is an excellent manuscript with a strong developmental biology focus, and its translational relevance is not immediately apparent; however, establishing such a link could significantly increase its impact. For example, the significance of these findings in ischemia-reperfusion injury, SOS/VOD, and sepsis could offer therapeutic avenues to stabilize endothelial function.

   The advance is the elegant discovery of a multifactorial endothelial-stabilizing mechanism in development, although its applicability to scenarios beyond developmental mutation remains unknown.

   The strengths are the clear and transparent experimental interrogation. Rightfully, the authors acknowledge that there would be a benefit in finalizing inflammatory blockade, genetic or antibody-mediated, to pin down the mechanistic circuit.

   The reviewer's expertise is: childhood liver diseases, developmental liver organoid generation, stem cells (iPSCs), cell reprogramming

   Reviewer #3

   Evidence, reproducibility and clarity:

   1. Wu et al. report antagonistic roles for chromatin remodelers Chd4 and Brg1, in endothelial cells, during liver development. There is a major flaw in the study which makes it difficult to interpret the conclusions. The genotypes of the mouse models used are flawed. The comparison should be made between two single knockouts (Chd4 single, Brg1 single), double mutants (Chd4/Brg1) and proper controls. For both "single KO", one allele of the other gene is also deleted - Chd4 -Ecko has one allele of Brg1 deleted and vice versa. Also, the proper control should be Chd4 fl/flBrg1fl/fl without the Cre. Since 3 alleles (not just two that belong to the same gene) are deleted in a single knockout, it is impossible to assign the effect to one gene.

   We acknowledge the fact that the single Brg1 and Chd4 EC knockouts in this study each carry a heterozygous deletion allele for the other remodeler (exact genotypes are shown in Fig. 1A). The mating strategy that yielded these mutants was chosen for three reasons. First, we have found that genetic background influences the embryonic phenotypes of these chromatin remodeler mutants3. Moreover, embryonic development at the stages analyzed in this study occurs quickly and requires precise timing for comparative analysis between genotypes. Therefore, it is most rigorous to study littermates when comparing single- and double-mutant embryos for BRG1 and CHD4. To achieve this, we used Brg1fl/fl;Chd4fl/fl females rather than Brg1fl/+;Chd4fl/+ females for timed matings. Although the former females cannot produce single knockout embryos without a compound heterozygous allele of the other remodeler, these females allowed us to generate single- and double-knockouts at a rate of 1/8 embryos. If we had used Brg1fl/+;Chd4fl/+ females for timed matings, we would have been able to generate “clean” single mutants with wildtype alleles of the other remodeler, but the single- and double-knockout generation rate would have been 1/32 embryos. This would have been an impractical mutant generation rate for this study. Second, our prior research demonstrates that heterozygous deletion of Chd4 or Brg1 does not produce the liver phenotypes seen with the respective homozygous deletions2,3. Third, the complete lethality of Chd4-ECko (Brg1fl/+;Chd4fl/fl;VE-cadherin-Cre+) mutants in this study demonstrates that deleting one allele of Brg1 cannot rescue Chd4-related lethality.

   As for controls in this study, we saw no evidence of phenotypes or of any gene deletion in our Cre- embryos (either in this study or in previous ones analyzing similar phenotypes2,3). Therefore, we used Cre- embryos for controls because they were generated at a 1/2 rate by our timed matings, which boosted our output for analyses.

   Specific points

   1. Fig 2c Plaur transcript - no statistical comparison between 2nd and 4th column, Chd4 Ecko vs double mutant. If there is not statistical difference, does not explain the rescue in double mutants

   Thank you for the suggestion. We have included a comparison between Chd4-ECko and Brg1/Chd4-ECdko in our revised Fig 2C. The Kruskal-Wallis test showed a significant difference between the Chd4-Ecko and* Brg1/Chd4-ECdko*groups (p=0.016). This indicates that Plaur induction in Chd4-Ecko LSECs is rescued in Brg1/Chd4-ECdko LSECs.

   1. Fig 2e. Comparison should be made between Plg-/- Chd4 fl/fl and Plg-/- Chd4 fl/fl Cre, not other genotypes

   This experiment aims to determine whether different levels of plasminogen (Plg) reduction can rescue the lethality caused by Chd4 deletion. To do this, we set up the mating strategy shown in Fig. 2E to produce appropriate littermate controls and to compare lethality among Plg+/+;Chd4-ECko, Plg+/-;Chd4-ECko, and Plg-/-;Chd4-ECko embryos. This comparison would not have been possible with embryos generated only from mice on a Plg-/- background.

   1. Fig. 4. How does Chd4 or Brg1 activity in endothelial cells lead to Icam1 activation in epithelial cells?

   Since cytokines like IFNg, TNFa, and IL1b can induce ICAM-1 expression in hepatocytes10, we speculate that ICAM-1 expression in hepatoblasts (ECAD+ cells in Fig. 4D) was induced by the elevated TNFa and IL1b produced in Chd4-ECko livers (Fig. 3G).

   1. Mice used in Figure 5 are Cdf4 fl/+ and Cdf4 fl/fl, no Brg1 deletion. The authors improperly compare these to Chd4-Ecko which have one allele of Brg1 deleted. The rescue needs to be done in the same genotype Chd4-Ecko.

   Please note that data from Fig. 5 were generated from cultured ECs (MS1 cells).

   Significance

   Wu et al. report antagonistic roles for chromatin remodelers Chd4 and Brg1, in endothelial cells, during liver development. There is a major flaw in the study which makes it difficult to interpret the conclusions. Genotypes that were chosen for the study make the data not interpretable

   Please see our response to your Question #1

   ---

   In summary, we have included the following changes to this revised manuscript:

   • New Figure 1B: Representative images and lethality rates for control, Chd4-ECko, Brg1-ECko, and Brg1/Chd4-ECdko embryos at E17.5.
   • New Figure 2C: qRT-PCR analysis of Chd4,* Brg1*, and Plaur gene transcripts in E12.5 control and mutant LSECs.
   • Regraphing of Figure 3G: qRT-PCR analysis of Tnf, Il6, and Il1b gene transcripts in E14.5 control and mutant livers.
   • New Figure S1: Representative images and lethality rates for control, Chd4fl/+;Vav-Cre+, and Chd4fl/fl;Vav-Cre+embryos at E18.5. References for this revision:

   Alva JA, Zovein AC, Monvoisin A, Murphy T, Salazar A, Harvey NL, Carmeliet P, Iruela-Arispe ML. VE-Cadherin-Cre-recombinase Transgenic Mouse: A Tool for Lineage Analysis and Gene Deletion in Endothelial Cells. Dev Dyn. 2006;235:759-767. doi: 10.1002/dvdy.20643 Crosswhite PL, Podsiadlowska JJ, Curtis CD, Gao S, Xia L, Srinivasan RS, Griffin CT. CHD4-regulated plasmin activation impacts lymphovenous hemostasis and hepatic vascular integrity. J Clin Invest. 2016;126:2254-2266. doi: 10.1172/JCI84652 Wu ML, Wheeler K, Silasi R, Lupu F, Griffin CT. Endothelial Chromatin-Remodeling Enzymes Regulate the Production of Critical ECM Components During Murine Lung Development. Arterioscler Thromb Vasc Biol. 2024;44:1784-1798. doi: 10.1161/ATVBAHA.124.320881 Shiojiri N, Inujima S, Ishikawa K, Terada K, Mori M. Cell lineage analysis during liver development using the spfash-heterozygous mouse. Lab Invest. 2001;81:17-25. doi: 10.1038/labinvest.3780208 Soares-da-Silva F, Peixoto M, Cumano A, Pinto-do OP. Crosstalk Between the Hepatic and Hematopoietic Systems During Embryonic Development. Front Cell Dev Biol. 2020;8:612. doi: 10.3389/fcell.2020.00612 Ema H, Nakauchi H. Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood. 2000;95:2284-2288. Kieusseian A, Brunet de la Grange P, Burlen-Defranoux O, Godin I, Cumano A. Immature hematopoietic stem cells undergo maturation in the fetal liver. Development. 2012;139:3521-3530. doi: 10.1242/dev.079210 Freitas-Lopes MA, Mafra K, David BA, Carvalho-Gontijo R, Menezes GB. Differential Location and Distribution of Hepatic Immune Cells. Cells. 2017;6. doi: 10.3390/cells6040048 Christensen JL, Wright DE, Wagers AJ, Weissman IL. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS Biol. 2004;2:E75. doi: 10.1371/journal.pbio.0020075 Satoh S, Nussler AK, Liu ZZ, Thomson AW. Proinflammatory cytokines and endotoxin stimulate ICAM-1 gene expression and secretion by normal human hepatocytes. Immunology. 1994;82:571-576.

   Read the original source
2. 

   Review Commons

   Jan 15, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   Wu et al. report antagonistic roles for chromatin remodelers Chd4 and Brg1, in endothelial cells, during liver development. There is a major flaw in the study which makes it difficult to interpret the conclusions. The genotypes of the mouse models used are flawed. The comparison should be made between two single knockouts (Chd4 single, Brg1 single), double mutants (Chd4/Brg1) and proper controls. For both "single KO", one allele of the other gene is also deleted - Chd4 -Ecko has one allele of Brg1 deleted and vice versa. Also, the proper control should be Chd4 fl/flBrg1fl/fl without the Cre. Since 3 alleles (not just two that belong …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   Wu et al. report antagonistic roles for chromatin remodelers Chd4 and Brg1, in endothelial cells, during liver development. There is a major flaw in the study which makes it difficult to interpret the conclusions. The genotypes of the mouse models used are flawed. The comparison should be made between two single knockouts (Chd4 single, Brg1 single), double mutants (Chd4/Brg1) and proper controls. For both "single KO", one allele of the other gene is also deleted - Chd4 -Ecko has one allele of Brg1 deleted and vice versa. Also, the proper control should be Chd4 fl/flBrg1fl/fl without the Cre. Since 3 alleles (not just two that belong to the same gene) are deleted in single knockout it is impossible to assign the effect on one gene.

   Specific points

   1. Fig 2c Plaur transcript - no statistical comparison between 2nd and 4th column, Chd4 Ecko vs double mutant. If there is not statistical difference, does not explain the rescue in double mutants
   2. Fig 2e. Comparison should be made between Plg-/- Chd4 fl/fl and Plg-/- Chd4 fl/fl Cre, not other genotypes
   3. Fig. 4. How does Chd4 or Brg1 activity in endothelial cells lead to Icam1 activation in epithelial cells?
   4. Mice used in Figure 5 are Cdf4 fl/+ and Cdf4 fl/fl, , no Brg1 deletion. The authors improperly compare these to Chd4-Ecko which have one allele of Brg1 deleted. The rescue need s to be done in the same genotype Chd4-Ecko.

   Significance

   Wu et al. report antagonistic roles for chromatin remodelers Chd4 and Brg1, in endothelial cells, during liver development. There is a major flaw in the study which makes it difficult to interpret the conclusions. Genotypes that were chosen for the study make the data not interpretable

   Read the original source
3. 

   Review Commons

   Jan 15, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   In essence, Wu et Al find that Chd4 mutant mice exhibit embryonic liver degeneration due to uPA-mediated plasmin hyperactivity and an ICAM-1-driven hyperinflammation and that additional mutation of BRG1 opposes this liver degeneration, possibly via ICAM-1.

   Generally, this is an excellent manuscript with a very logical sequence of experiments, although it has shortcomings such as validating their findings in an independent system, ideally human, and further establishing the translational relevance. Establishing translational relevance through mechanistic experiments that identify specific inflammatory tissue pathways, such as by …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   In essence, Wu et Al find that Chd4 mutant mice exhibit embryonic liver degeneration due to uPA-mediated plasmin hyperactivity and an ICAM-1-driven hyperinflammation and that additional mutation of BRG1 opposes this liver degeneration, possibly via ICAM-1.

   Generally, this is an excellent manuscript with a very logical sequence of experiments, although it has shortcomings such as validating their findings in an independent system, ideally human, and further establishing the translational relevance. Establishing translational relevance through mechanistic experiments that identify specific inflammatory tissue pathways, such as by blocking ICAM-1 and TNF-alpha, could also define developmental aberrations as a model for broader (patho)physiology and thereby enhance the impact on the field.

   Major

   • The embryonic and postnatal survival data of Chd4-ECko and Brg1/Chd4-Ecdko mice should be included in Fig. 1
   • What is the impact of Chd4-ECko and Brg1/Chd4-ECdko on the multicellular microenvironment? At a minimum, IF or spatial transcriptomics for hepatocyte and biliary markers, pericytes, and other mesenchymal cells would be recommended. Can there be a distinction made on what type of endothelial cell is affected? (sinusoidal lineage, vs. venous vs. lymphatic)
   • The experiments showing how endothelial Chd4 loss leads to a hyperinflammatory endothelial-and potentially hepatoblast-state are important. However, the relevance of immune cell infiltration in the hematopoietic-developing liver remains unclear. Which immune cells are presumably recruited to inflame the microenvironment then? Bone-marrow-derived? This aspect would benefit from experimental clarification, for example, using migration and/or direct co-culture versus indirect cell co-culture-ideally with or without ICAM-1 blockade-in vitro assays to determine if direct crosstalk with the CD45+ immune cell compartment explains the hyperinflammatory endothelia phenotype.
   • Related to the previous comment: Can the authors validate their findings in an independent, ideally human, cell-based system?
   • Identifying the specific hematopoietic/immune subset could further increase the paper's impact, as it would more definitively clarify the mechanism in the developing endothelial niche.
   • Also, can the authors show experimentally whether, conversely, Chd4 overexpression can limit an endothelial-type of inflammatory liver injury?

   Minor

   • A separate figure panel for Chd4fl/fl; Vav-Cre+ appears reasonable, instead of being shown as a table.

   Significance

   Generally, this is an excellent manuscript with a strong developmental biology focus, and its translational relevance is not immediately apparent; however, establishing such a link could significantly increase its impact. For example, the significance of these findings in ischemia-reperfusion injury, SOS/VOD, and sepsis could offer therapeutic avenues to stabilize endothelial function.

   The advance is the elegant discovery of a multifactorial endothelial-stabilizing mechanism in development, although its applicability to scenarios beyond developmental mutation remains unknown.

   The strengths are the clear and transparent experimental interrogation. Rightfully, the authors acknowledge that there would be a benefit in finalizing inflammatory blockade, genetic or antibody-mediated, to pin down the mechanistic circuit.

   The reviewer's expertise is: childhood liver diseases, developmental liver organoid generation, stem cells (iPSCs), cell reprogramming

   Read the original source
4. 

   Review Commons

   Jan 15, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Wu and Griffin describes a mechanism where CHD4 and BRG1, two chromatin remodelling enzymes, have antagonistic functions to regulate extracellular matrix (ECM) plasmin activity and sterile inflammatory phenotype in the endothelial cells of the developing liver. As a follow up from a previous study, the authors investigate the phenotype of embryonic-lethal endothelial-specific CHD4-knockout, leading to liver phenotype and embryo death, and the rescue of this phenotype when subsequently BRG1 is knocked-out also in the endothelium. First, the authors show that the increase in plasmin activator uPAR (which leads to ECM …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Wu and Griffin describes a mechanism where CHD4 and BRG1, two chromatin remodelling enzymes, have antagonistic functions to regulate extracellular matrix (ECM) plasmin activity and sterile inflammatory phenotype in the endothelial cells of the developing liver. As a follow up from a previous study, the authors investigate the phenotype of embryonic-lethal endothelial-specific CHD4-knockout, leading to liver phenotype and embryo death, and the rescue of this phenotype when subsequently BRG1 is knocked-out also in the endothelium. First, the authors show that the increase in plasmin activator uPAR (which leads to ECM degradation) in CHD4-KO embryos can be rescued by BRG1-KO, and that both CHD4 and BRG1 interact with the uPAR promoter. However, the authors demonstrate that reducing plasminogen by genetic knockout is unable to rescue the CHD4-KO embryos alone, suggesting an additional mechanism. By RNAseq analysis, the authors identify sterile inflammation as another potential contributor to the lethal phenotype of CHD4-KO embryos through increased expression of ICAM-1 in endothelial cells, also showing binding of both chromatin remodellers to ICAM-1 promoter. Finally, the authors use nonsteroidal anti-inflammatory drug carprofen, alone or in combination with plasminogen genetic knockout, and demonstrate CHD4-KO lethal embryonic phenotype rescue with the combination of plasminogen reduction and inflammation reduction, highlighting the synergistic role of both ECM degradation and sterile inflammation in this genetic KO.

   The findings of the manuscript are interesting, experiments well controlled and paper well written. While the work is of potential specialist interest to the field of liver development, there are several issues which authors should address before this paper can be published:

   Major issues:

   • The authors still see embryonic lethality of some embryos with endothelial BRG1-KO or combined endothelial CHD4/BRG1-KO - could the authors please show or at least comment in the discussion why those animals are dying?
   • In the qRT-PCR results Fig.2c, what is each dot?
   • In the same figure, I would expect that in CHD4-KO there is no CHD4 transcript, and in BRG1-KO there is no BRG1 transcript, rather than the reduction shown, which seems quite noisy (though significant) - is it this a result of normalisation? Or is indeed only a certain amount of the transcript reduced?
   • In the same figure, is the statistical testing performed before or after normalisation? This can introduce errors if done after normalisation.
   • In some cases, the authors show immunofluorescence images but do not specify how many biological replicates this represents (e.g. Fig.1d, 4c-d). This should be added.
   • I also encourage the authors to present a supplementary figure with at least one other biological replicate shown for imaging data (optional).
   • The plasminogen reduction by genetic modulation results in drastic changes to the embryos' appearance - is this a whole embryo KO or endothelial-specific KO? Can authors at least comment on the differences?
   • In Fig.2b, do I understand correctly only 1 sample was analysed with different areas plotted on the graph? If so, this experiment should be repeated on another set of embryos to be robust, and data plotted as a mean of each embryo (rather than areas).
   • Also in some graphs, authors specify that it was more than n>x embryos, but then - what are the dots on the graph representing? Each embryo? This should be specified (e.g. Fig.2b-c, but please check this in all the figure legends).
   • "we found Plaur was the only gene that was induced in CHD4-ECko LSECs at E12.5 (Figure S3D)." - I am not sure this is correct, as gene Plau is also increased in 2/3 samples?
   • I find the title and the running title somewhat misleading and too broad; the authors should specify more detail in the title about the content of the paper - the current statement of the title is somewhat true but shown only for one genetic model and not confirmed for all types of "lethal embryonic liver degeneration".

   Minor issues:

   • If an animal licence was used, its number should be specified in the ethics or methods section
   • In fig.3g it is very hard to see each of the samples, could authors try to improve this graph for clarity using colours-or split Y axis - or both?
   • "This indicates that ECs can play a pro-inflammatory role in embryonic livers and highlights the need for tight regulation to ensure normal liver growth." This sentence for me is misleading, EC are producing inflammatory signals only during the CHD4-KO according to the author's data, and authors do not show such data in normal homeostasis condition. Actually, the pro-inflammatory role here seems detrimental, and ECs should not exhibit it for correct development. The authors should rephrase this to be clearer.

   Significance

   General assessment: The study is well controlled and well written. The findings are interesting. The limitation of the findings is only 1 combination genetic model being studied, and it is unclear if the synergistic effect of sterile inflammation and ECM degradation is broadly applicable to other models, where embryo dies because of liver failure.

   Advance: The study makes an incremental advance, following up findings from a previous study. However, it is conceptually interesting.

   Audience: The audience for this manuscript would be a liver development specialist. However, broader concepts could also be applicable to liver disease.

   Expertise: I research in the field of liver regeneration and disease.

   Read the original source
5. 

   Version published to 10.1101/2025.09.16.676624 on bioRxiv

   Sep 18, 2025