A Novel 3D Visualization Method in Mice Identifies the Periportal Lamellar Complex (PLC) as a Key Regulator of Hepatic Ductal and Neuronal Branching Morphogenesis

  1. Tongtong Xu
  2. Fujun Cao
  3. Ruihan Zhou
  4. Qin Chen
  5. Jian Zhong
  6. Yulin Wang
  7. Chaoxin Xiao
  8. Banglei Yin
  9. Chong Chen
  10. Chengjian Zhao

  • Curated by eLife

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    eLife Assessment

    This study presents an important methodological advance-Liver-CUBIC combined with multicolor metallic nanoparticle perfusion-that enables high-resolution 3D visualization of the liver's complex multi-ductal architecture. The identification of the Periportal Lamellar Complex (PLC) as a novel perivascular structure with distinct cellular composition and low-permeability characteristics is convincing, supported by rigorous imaging data. The observed scaffolding role during fibrosis offers intriguing biological insights, though the functional claims would benefit from direct experimental validation.

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Abstract

The liver is a complex organ responsible for multiple functions, including metabolism, energy storage, detoxification, bile secretion, and immune regulation. Its highly organized vascular system plays a crucial role in maintaining functional zonation and tissue homeostasis. Within the liver, the hepatic artery, portal vein, hepatic vein, bile duct, and nerve networks intertwine to form an intricate three-dimensional architecture; however, traditional two-dimensional imaging fails to reveal their true spatial relationships, and current three-dimensional imaging methods remain insufficient to capture fine structural details. To achieve comprehensive visualization of these multi-ductal systems, we established a high-resolution three-dimensional imaging platform that combines multicolor perfusion of metallic compound nanoparticles (MCNPs) with an optimized tissue-clearing protocol (Liver-CUBIC), enabling simultaneous 3D reconstruction of the portal vein, hepatic artery, bile duct, and hepatic vein in mouse livers. Based on these data, we identified and defined a previously unrecognized structure located in the outer layer of the portal vein, termed the Periportal Lamellar Complex (PLC). The PLC encircles the portal vein between the vascular endothelium and the perisinusoidal region, exhibits low-permeability barrier characteristics, and contains a distinctive population of CD34⁺Sca-1⁺ endothelial cells. During liver fibrosis, the PLC extends from the portal vein toward the hepatic lobule, forming a structural scaffold that guides bile duct and nerve migration.

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  1. eLife

    eLife Assessment

    This study presents an important methodological advance-Liver-CUBIC combined with multicolor metallic nanoparticle perfusion-that enables high-resolution 3D visualization of the liver's complex multi-ductal architecture. The identification of the Periportal Lamellar Complex (PLC) as a novel perivascular structure with distinct cellular composition and low-permeability characteristics is convincing, supported by rigorous imaging data. The observed scaffolding role during fibrosis offers intriguing biological insights, though the functional claims would benefit from direct experimental validation.

  2. eLife

    Reviewer #1 (Public review):

    [Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the minor comments raised in the previous round of review.]

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Comments on revisions:

    The authors very nicely addressed all concerns from this reviewer. There are no further concerns and comments.

  3. eLife

    Reviewer #3 (Public review):

    Xu, Cao and colleagues aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the adult liver portal veins.
    Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting mRNA presence of genes linked to neurodevelopment, bile acid transport, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

    The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists. The importance of the CD34+/Sca1+ endothelial cell population and claims based on transcriptomic re-analysis require future assessment by functional experimental approaches to decipher the functional molecules involved in PLC formation, maintenance, and the involvement in injury response before establishing their role in biliary, arterial, and neural liver systems.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.
    This work proposes a new morphological feature of adult liver facilitating interaction between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - PLCs.

    Weaknesses:

    The importance of CD34+Sca1+ endothelial cell sub-population for PLC formation and function was not tested and warrants further validation.

  4. eLife

    Author Response:

    The following is the authors’ response to the previous reviews

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Comments on revisions:

    The authors very nicely addressed all concerns from this reviewer. There are no further concerns and comments.

    We thank the reviewer for the positive evaluation and helpful feedback.

    Reviewer #3 (Public review):

    Xu, Cao and colleagues aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the adult liver portal veins.

    Using available scRNAseq data, the authors assessed the CD34+/Sca-1+ cells' expression profile, highlighting mRNA presence of genes linked to neurodevelopment, bile acid transport, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

    The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists. The importance of the CD34+/Sca1+ endothelial cell population and claims based on transcriptomic re-analysis require future assessment by functional experimental approaches to decipher the functional molecules involved in PLC formation, maintenance, and the involvement in injury response before establishing their role in biliary, arterial, and neural liver systems.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.

    This work proposes a new morphological feature of adult liver facilitating interaction between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - PLCs.

    Weaknesses:

    The importance of CD34+Sca1+ endothelial cell sub-population for PLC formation and function was not tested and warrants further validation.

    We thank the reviewer for the valuable comment regarding the potential role of the CD34+/Sca-1+ endothelial cell sub-population in PLC function.

    We agree that direct functional validation would be a crucial next step to confirm the contribution of this specific sub-population to PLC formation and function. The focus of the present study remains on the spatial localization and reproducible characterization of PLC structures based on 3D imaging, as well as the relevant transcriptional features revealed by single-cell analysis.

    To avoid overinterpretation, we have revised the Discussion section accordingly, providing a more focused and cautious description of the related findings.

    Comments on revisions:

    I appreciate the author's effort to revise the text so it more rigorously adheres to the presented evidence. Following a thorough read of the revised text, a few remaining minor issues were identified in the Discussion.

    (1) From where comes the hard evidence for PLC being the stem cell niche in the following sentence?

    for the two following statements:

    This suggests that the PLC may not only provide structural support but also serve as a perivascular stem cell niche specific to the portal region, potentially involved in hematopoiesis and tissue regeneration.

    The PLC serves as a directional scaffold for ductal growth, a specialized stem cell niche, and a potential site of neurovascular coupling.

    We thank the reviewer for this important comment. We agree that the term “stem cell niche” may imply functional evidence for direct stem cell regulation, which was not demonstrated in this study. Our conclusions were based on the spatial enrichment and transcriptional features of CD34+/Sca-1+ endothelial populations expressing hematopoiesis-related genes in the portal region.

    To avoid overinterpretation, we have revised the sentence to remove the term “stem cell niche” and instead describe the PLC as being enriched in perivascular endothelial cell populations with hematopoiesis-related gene expression features. The revised text now reads:

    “These results suggest that, beyond structural support, the PLC in the portal region is enriched with perivascular endothelial cell populations exhibiting hematopoiesis-related gene expression features.”

    We have also modified the corresponding statement later in the Discussion. It now reads:

    “The PLC serves as a directional scaffold for ductal growth, displays distinct perivascular endothelial transcriptional features in the portal region, and may represent a potential site of neurovascular coupling.”

    We believe this wording more accurately reflects the descriptive and transcriptomic nature of our data without implying functional niche activity.

    (2) In the following paragraph, I lack references to the previously published evidence of liver innervation guidance mechanisms, such as the mesenchyme-mediated guidance (CD31- population) Gannoun et al., 2023 https://doi.org/10.1242/dev.201642, an important context for your finding.

    Further analysis showed significant upregulation of genes involved in neurodevelopment and axonal guidance in the CD34+/Sca-1+ cluster, along with activation of neuronal signaling pathways. Immunostaining confirmed the presence of TH+ sympathetic nerve fibers wrapping around the PLC in a "beads-on-a-string" pattern (Fig. 6), consistent with a classic neurovascular unit(Adori et al., 2021). Previous studies have shown that sympathetic nerves enter the liver along collagen fibers of Glisson's capsule and interact with hepatic arteries, portal veins, and bile duct epithelium, supporting the PLC as a scaffold for intrahepatic neurovascular integration.

    We thank the reviewer for highlighting the importance of previously published evidence regarding liver innervation guidance mechanisms. We agree that these studies provide important context for interpreting the neurodevelopmental and axon guidance–related transcriptional signatures observed in our dataset. Accordingly, we have revised the Discussion section to incorporate reference to mesenchyme-mediated axon guidance mechanisms in the portal region during liver development (Gannoun et al., 2023). This addition better situates our findings within the existing literature.

    (3) Several sentences have issues with a lack of space between words.

    We have carefully re-examined the entire manuscript for spacing and formatting inconsistencies and corrected minor typographical issues to ensure uniform formatting throughout the text.

  5. Version published to 10.7554/elife.108669.3 on eLife
  6. Version published to 10.7554/elife.108669 on eLife
  7. eLife

    eLife Assessment

    This study presents an important methodological advance-Liver-CUBIC combined with multicolor metallic nanoparticle perfusion-that enables high-resolution 3D visualization of the liver's complex multi-ductal architecture. The identification of the Periportal Lamellar Complex (PLC) as a novel perivascular structure with distinct cellular composition and low-permeability characteristics is convincing, supported by rigorous imaging data. The observed scaffolding role during fibrosis offers intriguing biological insights, though the functional claims would benefit from direct experimental validation.

  8. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34+Sca-1+ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Comments on revisions:

    The authors very nicely addressed all concerns from this reviewer. There are no further concerns and comments.

  9. eLife

    Reviewer #3 (Public review):

    Xu, Cao and colleagues aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the adult liver portal veins.
    Using available scRNAseq data, the authors assessed the CD34+Sca-1+ cells' expression profile, highlighting mRNA presence of genes linked to neurodevelopment, bile acid transport, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

    The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists. The importance of the CD34+/Sca1+ endothelial cell population and claims based on transcriptomic re-analysis require future assessment by functional experimental approaches to decipher the functional molecules involved in PLC formation, maintenance, and the involvement in injury response before establishing their role in biliary, arterial, and neural liver systems.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.
    This work proposes a new morphological feature of adult liver facilitating interaction between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - PLCs.

    Weaknesses:

    The importance of CD34+Sca1+ endothelial cell sub-population for PLC formation and function was not tested and warrants further validation.

    Comments on revisions:

    I appreciate the author's effort to revise the text so it more rigorously adheres to the presented evidence. Following a thorough read of the revised text, a few remaining minor issues were identified in the Discussion.

    (1) From where comes the hard evidence for PLC being the stem cell niche in the following sentence?
    for the two following statements:

    This suggests that the PLC may not only provide structural support but also serve as a perivascular stem cell niche specific to the portal region, potentially involved in hematopoiesis and tissue regeneration.

    The PLC serves as a directional scaffold for ductal growth, a specialized stem cell niche, and a potential site of neurovascular coupling.

    (2) In the following paragraph, I lack references to the previously published evidence of liver innervation guidance mechanisms, such as the mesenchyme-mediated guidance (CD31- population) Gannoun et al., 2023 https://doi.org/10.1242/dev.201642, an important context for your finding.

    Further analysis showed significant upregulation of genes involved in neurodevelopment and axonal guidance in the CD34+Sca-1+ cluster, along with activation of neuronal signaling pathways. Immunostaining confirmed the presence of TH+ sympathetic nerve fibers wrapping around the PLC in a "beads-on-a-string" pattern (Fig. 6), consistent with a classic neurovascular unit(Adori et al., 2021). Previous studies have shown that sympathetic nerves enter the liver along collagen fibers of Glisson's capsule and interact with hepatic arteries, portal veins, and bile duct epithelium, supporting the PLC as a scaffold for intrahepatic neurovascular integration.

    (3) Several sentences have issues with a lack of space between words.

  10. eLife

    Author response:

    The following is the authors’ response to the previous reviews

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34+Sca-1+ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Comments on revisions:

    The authors very nicely addressed all concerns from this reviewer. There are no further concerns or comments.

    We sincerely thank the reviewer for the positive evaluation of the revised manuscript.

    Reviewer #2 (Public review):

    Summary:

    The present manuscript of Xu et al. reports a novel clearing and imaging method focusing on the liver. The Authors simultaneously visualized the portal vein, hepatic artery, central vein, and bile duct systems by injected metal compound nanoparticles (MCNPs) with different colors into the portal vein, heart left ventricle, vena cava inferior and the extrahepatic bile duct, respectively. The method involves: trans-cardiac perfusion with 4% PFA, the injection of MCNPs with different colors, clearing with the modified CUBIC method, cutting 200 micrometer thick slices by vibratome, and then microscopic imaging. The Authors also perform various immunostaining (DAB or TSA signal amplification methods) on the tissue slices from MCNP-perfused tissue blocks. With the application of this methodical approach, the Authors report dense and very fine vascular branches along the portal vein. The authors name them as 'periportal lamellar complex (PLC)' and report that PLC fine branches are directly connected to the sinusoids. The authors also claim that these structures co-localize with terminal bile duct branches and sympathetic nerve fibers and contain endothelial cells with a distinct gene expression profile. Finally, the authors claim that PLC-s proliferate in liver fibrosis (CCl4 model) and act as scaffold for proliferating bile ducts in ductular reaction and for ectopic parenchymal sympathetic nerve sprouting.

    Strengths:

    The simultaneous visualization of different hepatic vascular compartments and their combination with immunostaining is a potentially interesting novel methodological approach.

    Weaknesses:

    This reviewer has some concerns about the validity of the microscopic/morphological findings as well as the transcriptomics results, and suggests that the conclusions of the paper may be critically viewed. Namely, at this point, it is still not fully clear that the 'periportal lamellar complex (PLC)' that the Authors describe really exists as a distinct anatomical or functional unit or these are fine portal branches that connect the larger portal veins into the adjacent sinusoid. Also, in my opinion, to identify the molecular characteristics of such small and spatially highly organized structures like those fine radial portal branches, the only way is to perform high-resolution spatial transcriptomics (instead of data mining in existing liver single cell database and performing Venn diagram intersection analysis in hepatic endothelial subpopulations). Yet, the existence of such structures with a distinct molecular profile cannot be excluded. Further research with advanced imaging and omics techniques (such as high resolution volume imaging, and spatial transcriptomics/proteomics) are needed to reproduce these initial findings.

    We thank the reviewer for the thoughtful and constructive comments. In response to the reviewer’s concerns regarding the anatomical and molecular definition of the periportal lamellar complex (PLC), we have further clarified the scope and methodological boundaries of the present study in the revised manuscript.

    Regarding the key question raised by the reviewer—namely, whether the PLC represents an independent anatomical or functional unit, or merely small portal venous branches connecting larger portal veins to adjacent sinusoids—we provide below a more detailed explanation of the criteria used to define the PLC in this study. The identification of the PLC is primarily based on periportal structures that can be reproducibly recognized by three-dimensional imaging across multiple mice, exhibiting a relatively consistent spatial distribution within the periportal region. The PLC could be stably observed across different MCNP dye color assignments and independent experimental batches. In addition, three-dimensional CD31 immunofluorescence consistently revealed vascular-associated signal distributions in the same periportal region, indirectly supporting its spatial association with the periportal vascular system.

    At the morphological level, the PLC appears as a periportal vasculature-associated structure distributed around the main portal vein trunk and maintains a relatively consistent spatial proximity to portal veins, bile ducts, and neural components in three-dimensional space. This highly conserved spatial organization across multiple tissue systems supports the anatomical positioning of the PLC as a relatively distinct structural tissue unit within the periportal region.

    The present study primarily focuses on a descriptive characterization of the three-dimensional anatomical organization and spatial relationships of the PLC based on volumetric imaging and vascular labeling strategies. As a complementary exploratory analysis, we reanalyzed endothelial cell populations potentially associated with the PLC using existing liver single-cell transcriptomic datasets. This analysis was intended to provide molecular-level information consistent with the structural observations and to offer preliminary clues to its potential biological functions, rather than to independently define the PLC at the spatial level or to functionally validate it.

    We fully acknowledge the value of spatial transcriptomic and spatial proteomic technologies in revealing molecular heterogeneity within tissue architecture. However, under current technical conditions, these approaches are largely dependent on thin tissue sections and are limited by spatial resolution and signal mixing effects, which still pose challenges for resolving periportal structures with pronounced three-dimensional continuity, such as the PLC. In the future, further integration of high-resolution volumetric imaging with spatial omics technologies may enable a more refined understanding of the molecular features and potential functions of the PLC at higher spatial resolution.

    Reviewer #3 (Public review):

    Summary:

    In the revised version of the manuscript authors addressed multiple comments, clarifying especially the methodological part of their work and PLC identification as a novel morphological feature of the adult liver portal veins. Tet is now also much clearer and has better flow.

    The additional assessment of the smartSeq2 data from Pietilä et al., 2025 strengthens the transcriptomic profiling of the CD34+Sca1+ cells and the discussion of the possible implications for the liver homeostasis and injury response. Why it may suffer from similar bias as other scRNA seq datasets - multiple cell fate signatures arising from mRNA contamination from proximal cells during dissociation, it is less likely that this would happen to yield so similar results.

    Nevertheless, a more thorough assessment by functional experimental approaches is needed to decipher the functional molecules and definite protein markers before establishing the PLC as the key hub governing the activity of biliary, arterial, and neuronal liver systems.

    The work does bring a clear new insight into the liver structure and functional units and greatly improves the methodological toolbox to study it even further, and thus fully deserves the attention of the Elife readers.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.

    This work proposes a new morphological feature of adult liver facilitating interaction between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - the Periportal Lamellar Complexes (PLCs).

    Weaknesses:

    The importance of CD34+Sca1+ endothelial cell subpopulation for PLC formation and function was not tested and warrants further validation.

    We thank the reviewer for the careful and constructive comments regarding the functional validation of cell populations associated with the PLC. The central aim of this study is to establish and validate a novel volumetric imaging and vascular labeling strategy and to apply it to the periportal region of the liver, thereby revealing previously underappreciated structural organizational patterns at the three-dimensional level, rather than to perform a systematic functional validation of specific cellular subpopulations.

    We agree that the precise roles of the CD34+Sca-1+ endothelial cell subpopulation in the formation and function of the periportal lamellar complex (PLC) have not been directly addressed through functional intervention experiments in the present study. Our conclusions are primarily based on three-dimensional imaging and spatial distribution analyses, which reveal a stable and consistent spatial association between this cell population and the PLC structure, but are not intended to independently support causal or functional inferences. The underlying functional mechanisms remain to be elucidated in future studies using genetic or functional perturbation approaches.

    In light of these considerations, we have further refined the relevant statements in the revised manuscript to more clearly define the functional scope and limitations of the current study in the Discussion section, and to avoid functional interpretations that extend beyond the direct support of the data. At the same time, we consider functional validation of the PLC to be an important and promising direction for future investigation.

    It should be emphasized that the present study is not primarily designed to provide direct functional validation, but rather to systematically characterize the three-dimensional structural features of the periportal lamellar complex (PLC) and its cellular associations using volumetric imaging and vascular labeling approaches. At this stage, we mainly provide spatial and histological evidence for the organizational relationship between the PLC structure and the CD34+Sca-1+ endothelial cell population, while their specific roles in PLC formation and functional regulation await further investigation.

    Recommendations for the authors:

    Reviewer #2 (Recommendations for the authors):

    I highly appreciate the Authors' endeavors to improve the manuscript. I am enlisting those points (from my original review) where I still have further comments.

    (2) I would suggest this sentence:

    "...the liver has evolved a highly complex and densely organized ductal vascular-neuronal network in the body, consisting primarily of the portal vein system, central vein system, hepatic artery system, biliary system, and intrahepatic autonomic nerve network [6, 7]."

    We thank the reviewer for the valuable suggestion. We have revised the relevant sentence accordingly, and the revised wording is as follows:

    “The liver has evolved a highly complex and densely organized vascular–biliary–neural network, primarily composed of the portal venous system, central venous system, hepatic arterial system, biliary system, and the intrahepatic autonomic neural network.”

    (3) I suggest renaming 'clearing efficiency' to 'clearing time', and revise the last sentence like:

    '...The results showed that the average transmittance increased by 20.12% in 1mm-thick cleared tissue slices.'

    We thank the reviewer for this helpful suggestion. Accordingly, we have replaced the term “clearing efficiency” with “clearing time” and revised the final sentence to reflect this change. The revised wording is as follows:

    “The results showed that the average transmittance increased by 20.12% in cleared tissue slices with a thickness of 1 mm.”

    (4) While the dye perfusion was indeed on full lobe, FigS1F also seems to be rather a thick section instead of a full 3d reconstruction. This is OK, but please, be clear and specific about this in the respective part of the ms.

    We thank the reviewer for the careful review and detailed comments. We would like to clarify that Fig. S1F shows whole-lobe imaging of the mouse left liver lobe obtained after dye perfusion at the whole-liver scale, rather than an image derived from a thick tissue section. Although this image does not represent a three-dimensional reconstruction, it does reflect imaging of the entire left liver lobe at the macroscopic level.

    In addition, for the reviewer’s reference, we have provided in this response a representative image of a 200 μm-thick liver tissue section to directly illustrate the morphological differences between thick-section imaging and whole-lobe imaging. We note that the third and fourth panels in Fig. 1G of the main text already show local imaging results from 200 μm-thick sections; in contrast, the comparative image provided here presents a larger field of view and overall morphology. To avoid redundancy, this additional image is included solely for clarification in the present response and has not been incorporated into the revised manuscript or the supplementary materials.

    (11) Regarding the 'transmission quantification':

    'Regarding the comparative quantification of different clearing methods, as the reviewer noted, nearly all aqueous or organic solvent based clearing techniques can achieve relatively uniform transparency in 1 mm thick tissue sections, so differences at this thickness are limited.'

    So, based on all these, I think, measuring/comparisons of clearing efficacy in the present form are kind of pointless --- one may consider omitting this part.

    We thank the reviewer for the valuable comments. The purpose of the transmittance quantification in this study was not to provide a comprehensive comparison among different tissue-clearing methods, but rather to serve as a quantitative reference supporting the optimization of the Liver-CUBIC protocol. Accordingly, we have narrowed and clarified the relevant statements in the revised manuscript to define their scope and avoid overinterpretation.

    The revised text now reads as follows:

    “Importantly, Liver-CUBIC treatment did not induce significant tissue expansion (Figure 1B–D). In addition, quantitative transmittance measurements in 1-mm-thick cleared tissue slices showed an average increase of 20.12% (P < 0.0001; 95% CI: 19.14–21.09; Figure 1E).”

    Author response image 1.

    (16) It is OK, but please, indicate this clearly in the Methods/Results because in its present form it may be confusing for the reader: which color means what.

    We thank the reviewer for this helpful request for clarification. We agree that the previous wording may have caused confusion regarding the meaning of different MCNP colors. Accordingly, we have revised the Methods section and the relevant figure legends to clearly state that the color assignment of MCNP dyes is not fixed across different experiments or figures. The use of different colors serves solely for visualization and presentation purposes, facilitating the distinction of anatomical structures in multichannel and three-dimensional imaging, and does not indicate any fixed or intrinsic correspondence between a specific color and a particular vascular or ductal system. We believe that this clarification will help prevent misinterpretation and improve the overall clarity of the manuscript.

    (17) Still I think the hepatic artery is extremely shrunk, while the portal vein is extremely dilated. Please, note that in the referring figure (from Adori et al), hepatic artery and portal vein are ca 50 micrometers and 250 micrometers in diameter, respectively. In your figure, as I see, ca. 9-10 micrometers and 125 micrometers, respectively. This means 5x (Adori) vs. 13-14x differences (you). I would not say that this is necessarily problematic --- but may reflect some perfusion issues that may be good to consider.

    We thank the reviewer for the careful comparison and acknowledge the quantitative differences pointed out. Compared with the study by Adori et al., the diameter ratio between the hepatic artery and the portal vein in our images does indeed differ to some extent. We believe that this discrepancy primarily arises from methodological differences in imaging and analysis strategies between the two studies.

    In the work by Adori et al., periportal vasculature identification and three-dimensional segmentation were mainly based on 488 nm autofluorescence signals acquired from inverted tissues. This signal predominantly reflects the overall outline of periportal tissue regions rather than direct imaging of the vascular lumen itself. Consequently, the measured “vessel diameter” largely represents a spatial domain delineated by surrounding periportal structures, and does not necessarily correspond to the actual or functional luminal diameter of the vessel.

    In contrast, the present study employed fluorescent MCNP dye perfusion under low perfusion pressure, combined with tissue clearing and three-dimensional optical imaging. Under these experimental conditions, the measured vessel diameters more closely reflect the perfusable luminal space of vessels in a fixed state, rather than their maximally dilated diameter, and are not defined by the morphology of surrounding tissues. This distinction is particularly relevant for the hepatic artery: as a high-resistance, smooth muscle–rich vessel, its diameter is highly sensitive to perfusion pressure and post-excision changes in vascular tone. In comparison, the portal vein exhibits greater compliance and is relatively less affected by these factors.

    Based on these methodological differences, the observation of relatively smaller apparent hepatic arterial diameters—and consequently a higher arterial-to-portal vein diameter ratio—under dye perfusion–based optical imaging conditions is an expected outcome. Importantly, the primary focus of the present study is the identification and characterization of the periportal lamellar complex (PLC) as a three-dimensional lamellar tissue structure that can be stably and reproducibly recognized across different samples and imaging conditions, rather than absolute comparisons of vascular diameters.

    (21) After the presented documentation, I still have some concerns that the 'periportal lamellar complex (PLC)' that the Authors describe is really a distinct anatomical or functional unit. The confocal panel in Fig. 4F is nice and high quality. However, as far as I see, it shows that CD34+/Sca-1+ immunostaining is not specific for the presumptive PLCs in the peri-portal region. Instead, Sca-1 immunoreactivity is highly abundant also in the midzone --- to which the supposed PLCs do not extend, according to the cartoon shown in panel D, same figure. Notably, this questions also the specificity of the single cell analysis.

    We thank the reviewer for this detailed and important comment regarding the specificity of CD34+/Sca-1+ markers and the definition of the periportal lamellar complex (PLC).

    It should be emphasized that the PLC is not defined on the basis of any single molecular marker, but rather by a reproducible periportal lamellar anatomical structure consistently revealed by three-dimensional imaging across multiple samples. The co-expression of CD34 and Sca-1 is interpreted within this clearly defined anatomical context and is used to characterize the molecular features of endothelial cells associated with the PLC structure.

    As shown in Fig. 4F, the co-expression of CD34 and Sca-1 delineates a continuous, lamellar endothelial structure surrounding the portal vein. In contrast, outside the periportal region—including the midlobular areas—Sca-1 or CD34 expression can also be detected, but these signals appear scattered and discontinuous, lacking an organized lamellar topology.

    In the single-cell transcriptomic analysis, we treated CD34+/Sca-1+ endothelial cells as an operational population to explore molecular features that may be enriched in the microenvironment of the periportal lamellar complex (PLC). Importantly, this analysis was intended to provide molecular clues associated with the PLC, rather than to precisely assign spatial locations or identities to individual cells.

    Occasional isolated Sca-1+ signals detected outside the periportal region do not affect the anatomical definition of the PLC, nor do they alter the interpretation of the single-cell analysis. These analyses serve to provide supportive and exploratory molecular information for the structural identification of the PLC, rather than constituting decisive spatial evidence.

    (23) '....In the manuscript, we have carefully stated that this analysis is exploratory in nature and have avoided overinterpretation. In future studies, high-resolution spatial omics approaches will be invaluable for more precisely delineating the molecular characteristics of these fine structures.'

    I do not find these statements either in the Discussion or in the Results. I must reiterate my opinion that the applied methodical approach in the single cell transcriptomics part has severe limitations, and the readers must be aware of this.

    We thank the reviewer for this further comment. We understand and acknowledge the reviewer’s concerns regarding the methodological limitations of single-cell transcriptomic analyses, and we agree that these limitations should be clearly communicated to readers in the main text.

    We acknowledge that in the previous version of the manuscript, the exploratory nature of the single-cell transcriptomic analysis and its methodological boundaries were discussed only in the response to reviewers and were not explicitly stated in the manuscript itself. We thank the reviewer for pointing out this omission. In the revised manuscript, we have now added explicit clarifications in the main text to prevent potential overinterpretation of these results.

    In the present study, our primary effort is focused on the descriptive characterization of the three-dimensional anatomical organization and spatial relationships of the PLC using volumetric imaging and vascular labeling strategies. As a complementary exploratory analysis, we reanalyzed existing liver single-cell transcriptomic datasets to examine endothelial cell populations exhibiting PLC-associated features, and performed differential gene expression and Gene Ontology enrichment analyses. Importantly, these results are intended to provide molecular-level support for the structural identification of the PLC and to offer preliminary insights into its potential biological functions. Accordingly, we have narrowed the presentation and interpretation of the single-cell analysis in both the Results and Discussion sections of the revised manuscript.

    In addition, we have expanded the Discussion to address the limitations of current spatial transcriptomic approaches in validating a continuous three-dimensional structure such as the PLC. Most existing spatial transcriptomic methods rely on two-dimensional tissue sections of 8–10 μm thickness, whereas identification of the PLC depends on three-dimensional imaging of tissue volumes with thicknesses of ≥200 μm, making reliable reconstruction of its spatial continuity from single sections challenging. Furthermore, because each spatial transcriptomic capture spot often encompasses multiple adjacent cells, signal mixing effects further limit precise resolution of specific periportal microstructures.

    Overall, we agree with the reviewer’s central point that the limitations of single-cell transcriptomic analyses should be clearly understood by readers. By explicitly clarifying the methodological boundaries and refining the related statements in the main text, we believe this concern has now been adequately addressed in the revised manuscript. We thank the reviewer for identifying this omission, which has helped to improve the rigor and clarity of the study.

    Reviewer #3 (Recommendations for the authors):

    (1) While interesting observations, suitable for discussion, the following sections are speculations, given that no functional characterization of PLC importance has been performed yet. This is the most felt when commenting on the role in hematopoiesis, which transiently takes place in the liver during embryogenesis (Khan et al 2016) but ceases to exist after ligation of the umbilical inlet. Adult Liver hematopoiesis remains controversial, and more solid evidence would need to be presented to support its existence in PLC regions.

    265 - These findings suggest that the Periportal Lamellar Complex (PLC) is not only a morphologically and spatially distinct, low-permeability vascular unit surrounding the portal vein, but also likely serves as a critical nexus connecting the portal vein, hepatic artery, and liver sinusoids. Thus, the PLC constitutes a key node within the interactive vascular network of the mouse liver.

    We thank the reviewer for the comments and suggestions regarding the potential functional interpretation of the periportal lamellar complex (PLC), particularly its possible association with hematopoietic function. We would like to clarify that the statement on page 265 was intended solely to describe the structural characteristics and spatial organization of the PLC within the periportal vascular network. Specifically, the original wording aimed to summarize the morphological features of the PLC and its spatial relationships among the portal vein, hepatic artery, and hepatic sinusoids.

    Nevertheless, to minimize potential misunderstanding, we have revised this section to avoid unnecessary functional implications. The revised text now reads:

    “These results suggest that the periportal lamellar complex (PLC) is a morphologically and spatially distinct vascular structure that surrounds the portal vein and may serve as a key organizational node coordinating the spatial relationships among the portal vein, hepatic artery, and hepatic sinusoids. Accordingly, the PLC represents an important structural element within the interactive vascular network of the mouse liver.”

    This revision preserves the structural significance of the PLC while avoiding overinterpretation of its functional roles.

    (2) The same is true also for this section, following Figure 3 - no functional experiment tested this. For example, diphtheria toxin is expressed in the CD34+Sca1+ population. Or at least a careful mapping of the developing liver, which would indicate if the PLC precedes or follows the BD development.

    356 as a spatial positional cue guiding bile duct growth and branching but also as a regulatory node involved in coordinating bile drainage from the hepatic lobule into the biliary network.

    To avoid potential misunderstanding, we have further refined and revised the statements in the manuscript regarding the functional interpretation of the periportal lamellar complex (PLC) and its relationship to bile duct development. We agree that cell ablation strategies are of great importance for functional validation studies. However, it should be noted that CD34 and Sca-1 are relatively broadly expressed markers during liver development, labeling multiple endothelial, mesenchymal, and progenitor cell populations, and their expression is not restricted to the PLC. Owing to this broad expression pattern, ablation of CD34+Sca-1+ cell populations would likely exert widespread effects on vascular and stromal structures, thereby complicating the distinction between direct PLC-specific effects and secondary developmental alterations. As such, this strategy may present technical limitations for specifically dissecting the role of the PLC in bile duct development. At the same time, given that the primary objective of this study is the systematic characterization of the three-dimensional anatomical features and spatial organization of the PLC, we have correspondingly revised the manuscript to restrict statements regarding the relationship between the PLC and bile ducts to spatial associations supported by the current data. Specifically, our results show that primary bile ducts run along the main portal vein trunk, secondary bile ducts exhibit directed branching toward the PLC region, and terminal bile duct branches tend to spatially cluster in the vicinity of the PLC, thereby forming a reproducible periportal spatial arrangement. Based on these observations, the PLC delineates a relatively conserved anatomical microenvironment within the portal region, whose spatial position is closely associated with the organization and terminal distribution of the intrahepatic bile duct network.

    We believe that these revisions more accurately reflect the experimental evidence and the defined scope of the present study.

    (3) The following statement ought to be rephrased or skipped, considering that CD34 and Sca1 (Ly6a) are markers of periportal endothelial cells (Pietilä et al., 2025, Gómez-Salinero et al., 2022) and as shown by the authors in their own Fig. 6D. In this context and the context of the CCL4 experiments, a "simple" proliferative progenitor portal vein endothelial cell phenotype, suggested also by the presence of DLL4 (Fig5A) and JAG1 (Pietilä et al., 2025) (Benedito et al., 2009) ought to be considered.

    409 Notably, CD34 and Sca-1 (Ly6a) were co-expressed exclusively within PLC structures surrounding the portal vein, but absent from central vein ECs and midzonal LSECs (Figure 4F).

    We thank the reviewer for pointing out the potential imprecision in this wording. We agree that both CD34 and Sca-1 (Ly6a) are well-established markers of periportal endothelial cells, as previously reported (Pietilä et al., 2025; Gómez-Salinero et al., 2022), and as also illustrated in Fig. 4F of our study.

    Accordingly, the original statement suggesting that CD34 and Sca-1 are co-expressed exclusively within the PLC structure may indeed represent an overinterpretation. Following the reviewer’s suggestion, we have revised the relevant text on page 409 by removing the exclusive phrasing (“only in”) and by emphasizing instead that CD34+Sca-1+ endothelial cells are enriched in periportal regions associated with the PLC, rather than being specific to or confined within the PLC.

    In addition, in the context of the CCl4-induced liver fibrosis model, we agree with the reviewer that the observed expression of DLL4 and JAG1 under fibrotic conditions is more appropriately interpreted as reflecting an activated or proliferative periportal endothelial progenitor–like phenotype, rather than defining a novel endothelial lineage. The corresponding statements in the revised manuscript have been adjusted accordingly.

    (4) Again, these concluding sentences are based on correlative evidence of mRNA expression and literature but not experimental evidence.

    436 These findings suggest that this unique endothelial cell subset in the periportal region may possess dual regulatory functions in both metabolic and hematopoietic modulation

    441 results suggest that PLC endothelial cells may not only regulate periportal microcirculatory blood flow but also help establish a specialized microenvironment that potentially supports periportal hematopoietic regulation, contributing to stem cell recruitment, vascular homeostasis, and tissue repair.

    We thank the reviewer for this thoughtful comment. We agree that these statements are primarily based on transcriptomic correlation analyses and support from previous literature, rather than direct functional experimental evidence.

    Accordingly, in the revised manuscript, we have appropriately toned down and adjusted the relevant concluding statements to more accurately reflect their inferential nature. The revised wording emphasizes associations and potential involvement, rather than definitive functional roles. These changes preserve the overall scientific interpretation while aligning the level of inference more closely with the available evidence.

    The revised text now reads:

    “Finally, we found that the main trunk of the PLC is primarily composed of CD34+Sca-1+CD31+ endothelial cells (Fig. 4J). These CD34+Sca-1+ double-positive cells are mainly distributed in the basal region of the PLC structure and exhibit molecular features associated with hematopoiesis. Taken together, these results suggest that PLC endothelial cells may contribute to the establishment of a local microenvironment related to periportal hematopoietic regulation and may play potential roles in stem cell recruitment and maintenance of vascular homeostasis.”

    (5) The following part is speculative and based on re-analysis from the dataset that was gathered after 6 more weeks of CCL4 treatment (12weeks Su et al., 2021), then in the linked experiments from the manuscript. And should be moved to discussion or removed.

    504 Moreover, single-cell transcriptomic re-analysis revealed significant upregulation of bile duct-related genes in the CD34+Sca-1+ endothelium of PLC in fibrotic liver, with notably high expression of Lgals1 (Galectin-1) and Hgf (Figure 5G). Previous studies have shown that Galectin-1 is absent in normal liver parenchyma but highly expressed in intrahepatic cholangiocarcinoma (ICC), correlating with tumor dedifferentiation and invasion (Bacigalupo, Manzi, Rabinovich, & Troncoso, 2013; Shimonishi et al., 2001). Additionally, hepatocyte growth factor (HGF), particularly in combination with epidermal growth factor (EGF) in 3D cultures, promotes hepatic progenitor cells to form bile duct-polarized cystic structures (N. Tanimizu, Miyajima, & Mostov, 2007). Together, these findings suggest the PLC endothelium may act as a key regulator of bile duct branching and fibrotic microenvironment remodeling in liver fibrosis.

    Collectively, our results demonstrate that the PLC, situated between the portal vein and periportal sinusoidal endothelium, constitutes a critical vascular microenvironmental unit. It may not only colocalize with bile duct branches under normal physiological conditions, but also through its basal CD34+Sca-1+ double-positive endothelial cells, potentially orchestrate bile duct epithelial proliferation, branching morphogenesis, and bile acid transport homeostasis via multiple signaling pathways. Particularly during liver fibrosis progression, the PLC exhibits dynamic structural extension, serving as a spatial scaffold facilitating terminal bile duct migration and expansion into the hepatic parenchyma (Figure 5H). These findings highlight the PLC endothelial cell population and the vascular-bile duct interface as key regulatory hubs in bile duct regeneration, tissue repair, and pathological remodeling, providing novel cellular and molecular insights for understanding bile duct-related diseases such as ductular reaction, cholangiocarcinoma, and cholestatic disorders, and offering potential targets for therapeutic intervention.

    We thank the reviewer for this careful and thought-provoking comment. We understand and agree with the reviewer’s assessment that this section involves a degree of inference, as the analysis is based on a re-analysis of a previously published single-cell transcriptomic dataset from a CCl4-induced liver fibrosis model (Su et al., 2021), rather than on experimental data directly generated in the present study.

    In response to the reviewer’s suggestion, we have carefully re-examined and revised the relevant paragraphs. Without altering the overall structure of the manuscript, we have appropriately moderated the wording to clarify that these results primarily describe the transcriptional features of PLC-associated CD34+Sca-1+ endothelial cells under fibrotic conditions, and their associations with bile duct–related gene expression, rather than providing direct functional evidence for their roles in bile duct branching or microenvironmental remodeling.

    In addition, we have explicitly clarified in the main text the data source and methodological limitations of the single-cell transcriptomic analysis, and emphasized that these findings should be interpreted in conjunction with the spatial information revealed by three-dimensional imaging. Through these revisions, we aim to retain the value of this analysis in providing complementary molecular insight into PLC characteristics, while avoiding potential over-interpretation of its functional implications.

    Formal suggestions:

    (6) The following sentence would benefit from being more clearly written.

    263 - The formation of PLC structures in the adventitial layer may participate in local blood flow regulation, maintenance of microenvironmental homeostasis.

    We thank the reviewer for this helpful suggestion. The sentence has been revised to improve clarity by correcting the parallel structure and refining the wording.

    The formation of PLC structures in the adventitial layer may participate in local blood flow regulation and the maintenance of microenvironmental homeostasis.

    (7) The following sentence is misleading as it implies cell sorting, and "subsetted" rather than "sorted" should be used.

    414 Based on this, we sorted CD34+Sca-1+ endothelial populations from the total liver EC pool (Figure 4G).

    Thank you for your comment.

    We have revised the term as suggested. This avoids the misleading implication of physical sorting, as our operation was analytical subsetting of the target subpopulation.

    We appreciate your careful review.

    (8) Correct typos, especially in the results section related to Fig. 6. and formatting issues in the discussion.

    730 Morphologically, the PLC shares features with previously described telocytes (TCs)- 731 a recently identified class of interstitial cells in the liver observed via transmission electron

    We thank the reviewer for pointing out this textual error. In the submitted version, the sentence describing the morphological similarity between the PLC and previously reported telocytes was inadvertently interrupted due to a punctuation issue. This has now been corrected to ensure sentence integrity and consistent formatting.

  11. Version published to 10.7554/elife.108669.2 on eLife
  12. eLife

    eLife Assessment

    This study uses a novel 3D imaging method to identify the Periportal Lamellar Complex (PLC), an important new structure. Although the methodological advancement and morphological descriptions are convincing, the evidence for its proposed function is incomplete, relying on transcriptomic correlation rather than direct experimental validation. The work would therefore be strengthened by focusing its claims on the robust methodological advancement and detailed morphological characterization.

  13. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Comments on revisions:

    The authors very nicely addressed all concerns from this reviewer. There are no further concerns or comments.

  14. eLife

    Reviewer #2 (Public review):

    Summary:

    The present manuscript of Xu et al. reports a novel clearing and imaging method focusing on the liver. The Authors simultaneously visualized the portal vein, hepatic artery, central vein, and bile duct systems by injected metal compound nanoparticles (MCNPs) with different colors into the portal vein, heart left ventricle, vena cava inferior and the extrahepatic bile duct, respectively. The method involves: trans-cardiac perfusion with 4% PFA, the injection of MCNPs with different colors, clearing with the modified CUBIC method, cutting 200 micrometer thick slices by vibratome, and then microscopic imaging. The Authors also perform various immunostaining (DAB or TSA signal amplification methods) on the tissue slices from MCNP-perfused tissue blocks. With the application of this methodical approach, the Authors report dense and very fine vascular branches along the portal vein. The authors name them as 'periportal lamellar complex (PLC)' and report that PLC fine branches are directly connected to the sinusoids. The authors also claim that these structures co-localize with terminal bile duct branches and sympathetic nerve fibers and contain endothelial cells with a distinct gene expression profile. Finally, the authors claim that PLC-s proliferate in liver fibrosis (CCl4 model) and act as scaffold for proliferating bile ducts in ductular reaction and for ectopic parenchymal sympathetic nerve sprouting.

    Strengths:

    The simultaneous visualization of different hepatic vascular compartments and their combination with immunostaining is a potentially interesting novel methodological approach.

    Weaknesses:

    This reviewer has some concerns about the validity of the microscopic/morphological findings as well as the transcriptomics results, and suggests that the conclusions of the paper may be critically viewed. Namely, at this point, it is still not fully clear that the 'periportal lamellar complex (PLC)' that the Authors describe really exists as a distinct anatomical or functional unit or these are fine portal branches that connect the larger portal veins into the adjacent sinusoid. Also, in my opinion, to identify the molecular characteristics of such small and spatially highly organized structures like those fine radial portal branches, the only way is to perform high-resolution spatial transcriptomics (instead of data mining in existing liver single cell database and performing Venn diagram intersection analysis in hepatic endothelial subpopulations). Yet, the existence of such structures with a distinct molecular profile cannot be excluded. Further research with advanced imaging and omics techniques (such as high resolution volume imaging, and spatial transcriptomics/proteomics) are needed to reproduce these initial findings.

  15. eLife

    Reviewer #3 (Public review):

    Summary:

    In the revised version of the manuscript authors addressed multiple comments, clarifying especially the methodological part of their work and PLC identification as a novel morphological feature of the adult liver portal veins. Tet is now also much clearer and has better flow.

    The additional assessment of the smartSeq2 data from Pietilä et al., 2025 strengthens the transcriptomic profiling of the CD34+Sca1+ cells and the discussion of the possible implications for the liver homeostasis and injury response. Why it may suffer from similar bias as other scRNA seq datasets - multiple cell fate signatures arising from mRNA contamination from proximal cells during dissociation, it is less likely that this would happen to yield so similar results.

    Nevertheless, a more thorough assessment by functional experimental approaches is needed to decipher the functional molecules and definite protein markers before establishing the PLC as the key hub governing the activity of biliary, arterial, and neuronal liver systems.

    The work does bring a clear new insight into the liver structure and functional units and greatly improves the methodological toolbox to study it even further, and thus fully deserves the attention of the Elife readers.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.

    This work proposes a new morphological feature of adult liver facilitating interaction between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - the Periportal Lamellar Complexes (PLCs).

    Weaknesses:

    The importance of CD34+Sca1+ endothelial cell subpopulation for PLC formation and function was not tested and warrants further validation.

  16. eLife

    Author response:

    The following is the authors’ response to the original reviews

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Weaknesses:

    This manuscript is well-written, organized, and informative. However, there are some points that need to be clarified.

    (1) After MCNP-dye injection, does it remain in the blood vessels, adsorb onto the cell surface, or permeate into the cells? Does the MCNP-dye have cell selectivity?

    The experimental results showed that after injection, the MCNP series nanoparticles predominantly remained within the lumens of blood vessels and bile ducts, with their tissue distribution determined by physical perfusion. No diffusion of the dye signal into the surrounding parenchymal tissue was observed, nor was there any evidence of adsorption onto the cell surface or entry into cells. The newly added Supplementary Figure S2A–H further confirmed this feature, demonstrating that the dye signals were strictly confined to the luminal space, clearly delineating the continuous course of blood vessels and the branching morphology of bile ducts. These findings strongly support the conclusion that “MCNP dyes are distributed exclusively within the luminal compartments.”

    Therefore, the MCNP dyes primarily serve as intraluminal tracers within the tissue rather than as labels for specific cell types.

    (2) All MCNP-dyes were injected after the mice were sacrificed, and the mice's livers were fixed with PFA. After the blood flow had ceased, how did the authors ensure that the MCNP-dyes were fully and uniformly perfused into the microcirculation of the liver?

    Thank you for the reviewer’s valuable comments. Indeed, since all MCNP dyes were perfused after the mice were euthanized and blood circulation had ceased, we cannot fully ensure a homogeneous distribution of the dye within the hepatic microcirculation. The vascular labeling technique based on metallic nanoparticle dyes used in this study offers clear imaging, stable fluorescence intensity, and multiplexing advantages; however, it also has certain limitations. The main issue is that the dye distribution within the hepatic parenchyma can be affected by factors such as lobular overlap, local tissue compression, and variations in vascular pathways, resulting in regional inhomogeneity of dye perfusion. This is particularly evident in areas where multiple lobes converge or where anatomical structures are complex, leading to local dye accumulation or over-perfusion.

    In our experiments, we attempted to minimize local blockage or over-perfusion by performing PBS pre-flushing and low-pressure, constant-speed perfusion. Nevertheless, localized dye accumulation or uneven distribution may still occur in lobe junctions or structurally complex regions. Such variation represents one of the methodological limitations. Overall, the dye signals in most samples remained confined to the vascular and biliary lumens, and the distribution pattern was highly reproducible.

    We have addressed this issue in the Discussion section but would like to emphasize here that, although this system has clear advantages, it remains sensitive to anatomical variability in the liver—such as lobular overlap and vascular heterogeneity. At vascular junctions, local perfusion inhomogeneity or dye accumulation may occur; therefore, injection strategies and perfusion parameters should be adjusted according to liver size and vascular condition to improve reproducibility and imaging quality. It should also be noted that the results obtained using this method primarily aim to visualize the overall and fine anatomical structures of the hepatic vascular system rather than to quantitatively reflect hemodynamic processes. In the future, we plan to combine in vivo perfusion or dynamic fluid modeling to further validate the diffusion characteristics of the dyes within the hepatic microcirculation.

    (3) It is advisable to present additional 3D perspective views in the article, as the current images exhibit very weak 3D effects. Furthermore, it would be better to supplement with some videos to demonstrate the 3D effects of the stained blood vessels.

    Thank you for the reviewer’s valuable comments. In response to the suggestion, we have added perspective-rendered images generated from the 3D staining datasets to provide a more intuitive visualization of the spatial morphology of the hepatic vasculature. These images have been included in Figure S2A–J. In addition, we have prepared supplementary videos (available upon request) that dynamically display the three-dimensional distribution of the stained vessels, further enhancing the spatial perception and visualization of the results.

    (4) In Figure 1-I, the authors used MCNP-Black to stain the central veins; however, in addition to black, there are also yellow and red stains in the image. The authors need to explain what these stains are in the legend.

    Thank you for the reviewer’s constructive comment. In Figure 1I, MCNP-Black labels the central vein (black), MCNP-Yellow labels the portal vein (yellow), MCNP-Pink labels the hepatic artery (pink), and MCNP-Green labels the bile duct (green). We have revised the Figure 1 legend to include detailed descriptions of the color signals and their corresponding structures to avoid any potential confusion.

    (5) There is a typo in the title of Figure 4F; it should be "stem cell".

    Thank you for the reviewer’s careful correction. We have corrected the spelling error in the title of Figure 4F to “stem cell” and updated it in the revised manuscript.

    (6) Nuclear staining is necessary in immunofluorescence staining, especially for Figure 5e. This will help readers distinguish whether the green color in the image corresponds to cells or dye deposits.

    We thank the reviewer for the valuable suggestion. We understand that nuclear staining can help determine the origin of fluorescence signals. However, in our three-dimensional imaging system, the deep signal acquisition range after tissue clearing often causes nuclear dyes such as DAPI to generate highly dense and widespread fluorescence, especially in regions rich in vascular structures, which can obscure the fine vascular and perivascular details of interest. Therefore, this study primarily focuses on high-resolution visualization of the spatial architecture of the vascular and biliary systems. We have added an explanation regarding this point in Figures S2I–J.

    Reviewer #2 (Public review):

    Summary:

    The present manuscript of Xu et al. reports a novel clearing and imaging method focusing on the liver. The authors simultaneously visualized the portal vein, hepatic artery, central vein, and bile duct systems by injecting metal compound nanoparticles (MCNPs) with different colors into the portal vein, heart left ventricle, inferior vena cava, and the extrahepatic bile duct, respectively. The method involves: trans-cardiac perfusion with 4% PFA, the injection of MCNPs with different colors, clearing with the modified CUBIC method, cutting 200 micrometer thick slices by vibratome, and then microscopic imaging. The authors also perform various immunostaining (DAB or TSA signal amplification methods) on the tissue slices from MCNP-perfused tissue blocks. With the application of this methodical approach, the authors report dense and very fine vascular branches along the portal vein. The authors name them as 'periportal lamellar complex (PLC)' and report that PLC fine branches are directly connected to the sinusoids. The authors also claim that these structures co-localize with terminal bile duct branches and sympathetic nerve fibers, and contain endothelial cells with a distinct gene expression profile. Finally, the authors claim that PLC-s proliferate in liver fibrosis (CCl4 model) and act as a scaffold for proliferating bile ducts in ductular reaction and for ectopic parenchymal sympathetic nerve sprouting.

    Strengths:

    The simultaneous visualization of different hepatic vascular compartments and their combination with immunostaining is a potentially interesting novel methodological approach.

    Weaknesses:

    This reviewer has several concerns about the validity of the microscopic/morphological findings as well as the transcriptomics results. In this reviewer's opinion, the introduction contains overstatements regarding the potential of the method, there are severe caveats in the method descriptions, and several parts of the Results are not fully supported by the documentation. Thus, the conclusions of the paper may be critically viewed in their present form and may need reconsideration by the authors.

    We sincerely thank the reviewer for the thorough evaluation and constructive comments on our study. We fully understand and appreciate the reviewer’s concerns regarding the methodological validity and interpretation of the results. In response, we have made comprehensive revisions and additions to the manuscript as follows:

    First, we have carefully revised the Introduction and Discussion sections to provide a more balanced description of the methodological potential, removing statements that might be considered overstated, and clarifying the applicable scope and limitations of our approach (see the revised Introduction and Discussion).

    Second, we have substantially expanded the Methods section with detailed information on model construction, imaging parameters, data processing workflow, and technical aspects of the single-cell transcriptomic reanalysis, to enhance the transparency and reproducibility of the study.

    Third, we have added additional references and explanatory notes in the Results section to better support the main conclusions (see Section 6 of the Results).

    Finally, we have rechecked and validated all experimental data, and conducted a verification analysis using an independent single-cell RNA-seq dataset (Figure S6). The results confirm that the morphological observations and transcriptomic findings are consistent and reproducible across independent experiments.

    We believe these revisions have greatly strengthened the reliability of our conclusions and the overall scientific rigor of the manuscript. Once again, we sincerely appreciate the reviewer’s valuable comments, which have been very helpful in improving the logic and clarity of our work.

    Reviewer #3 (Public review):

    Summary:

    In the reviewed manuscript, researchers aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the portal vein axis. The PLC originates from the portal vein and is characterized by a unique population of CD34⁺Sca-1⁺ dual-positive endothelial cells. Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting the mRNA presence of genes linked to neurodevelopment, biliary function, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

    The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver at the same time. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists; however, some claims need more thorough assessment by functional experimental approaches to decipher the functional molecules and the sequence of events before establishing the PLC as the key hub governing the activity of biliary, arterial, and neuronal liver systems. Similarly, the level of detail of the methods section does not appear to be sufficient to exactly recapitulate the performed experiments, which is of concern, given that the new technique is a cornerstone of the manuscript.

    Nevertheless, the work does bring a clear new insight into the liver structure and functional units and greatly improves the methodological toolbox to study it even further, and thus fully deserves the attention of readers.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.

    This work proposes a new biological framework between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - the Periportal Lamellar Complexes (PLCs).

    Weaknesses:

    Possible overinterpretation of the CD34+Sca1+ findings was built on re-analysis of one scRNAseq dataset.

    Lack of detail in the materials and methods section greatly limits the usefulness of the new technique to other researchers.

    We thank the reviewer for this important comment. We agree that when conclusions are mainly based on a single dataset, overinterpretation should be avoided. In response to this concern, we have carefully re-evaluated and clearly limited the scope of our interpretation of the scRNA-seq analysis. In addition, we performed a validation analysis using an independent single-cell RNA-seq dataset (see new Figure S6), which consistently confirmed the presence and characteristic transcriptional profile of the periportal CD34⁺Sca1⁺ endothelial cell population. These supplementary analyses strengthen the robustness of our findings and address the reviewer’s concern regarding potential overinterpretation.

    In the revised manuscript, we have also greatly expanded the Materials and Methods section by providing detailed information on sample preparation, imaging parameters, data processing workflow, and single-cell reanalysis procedures. These revisions substantially improve the transparency and reproducibility of our methodology, thereby enhancing the usability and reference value of this technique for other researchers.

    Recommendations for the authors:

    Reviewer #2 (Recommendations for the authors):

    Introduction

    (1) In general, the Introduction is very lengthy and repetitive. It needs extensive shortening to a maximum of 2 A4 pages.

    We thank the reviewer for the valuable suggestions. We have thoroughly condensed and restructured the Introduction, removing redundant content and merging related paragraphs to make the theme more focused and the logic clearer. The revised Introduction has been shortened to within two A4 pages, emphasizing the scientific question, innovation, and technical approach of the study.

    (2) Please correct this erroneous sentence:

    '...the liver has evolved the most complex and densely n organized vascular network in the body, consisting primarily of the portal vein system, central vein system, hepatic artery system, biliary system, and intrahepatic autonomic nerve network [6, 7].'

    We thank the reviewer for pointing out this spelling error. The revised sentence is as follows:

    “…the liver has evolved the most complex and densely organized ductal-vascular network in the body, consisting primarily of the portal vein system, central vein system, hepatic artery system, biliary system, and intrahepatic autonomic nerve network [6, 7].”

    (3) '...we achieved a 63.89% improvement in clearing efficiency and a 20.12% increase in tissue transparency'

    Please clarify what you exactly mean by 'clearing efficiency' and 'increased tissue transparency'.

    We thank the reviewer for the valuable comments and have clarified the relevant terminology in the revised manuscript.

    “Clearing efficiency” refers to the improvement in the time required for the liver tissue to become completely transparent when treated with the optimized Liver-CUBIC protocol (40% urea + H₂O₂), compared with the conventional CUBIC method. In this study, the clearing time was reduced from 9 days to 3.25 days, representing a 63.89% increase in time efficiency.

    “Tissue transparency” refers to the ability of the cleared tissue to transmit visible light. We quantified the optical transparency by measuring light transmittance across the 400–900 nm wavelength range using a microplate reader. The results showed that the average transmittance increased by 20.12%, indicating that Liver-CUBIC treatment markedly enhanced the optical clarity of the liver tissue.

    (4) I am concerned about claiming this imaging method as real '3D imaging'. Namely, while the authors clear full lobes, they actually cut the cleared lobes into 200-micrometer-thick slices and perform further microscopy imaging on these slices. Considering that they focus on ductular structures of the liver (such as vasculature, bile duct system, and innervations), 200 micrometer allows a very limited 3D overview, particularly in comparison with the whole-mount immuno-imaging methods combined with light sheet microscopy (such as Adori 2021, Liu 2021, etc). In this context, I feel several parts of the Introduction to be an overstatement: besides of emphasizing the advantages of the technique (such as simultaneous visualization of different hepatic vascular compartments and the bile duct system by MCNPs, the combination with immunostainings), the authors must honestly discuss the limitations (such as limited tissue overview, potential dye perfusion problems - uneven distribution of the dye etc).

    We appreciate the reviewer’s insightful comments. It is true that most of the imaging depth in this study was limited to approximately 200 μm, and thus it could not achieve whole-liver three-dimensional imaging comparable to light-sheet microscopy. However, the primary focus of our study was to resolve the microscopic intrahepatic architecture, particularly the spatial relationships among blood vessels, bile ducts, and nerve fibers. Through high-resolution imaging of thick tissue sections, combined with MCNP-based multichannel labeling and immunofluorescence co-staining, we were able to accurately delineate the three-dimensional distribution of these microstructures within localized regions.

    In addition to thick-section imaging, we also obtained whole-lobe dye perfusion data (as shown in Figure S1F), which comprehensively depict the three-dimensional branching patterns and distribution of the vascular systems within the liver lobe. These images were acquired from intact liver lobes perfused with MCNP dyes, revealing a continuous vascular network extending from major trunks to peripheral branches, thereby demonstrating that our approach is also capable of achieving organ-level visualization.

    We have added this image and a corresponding description in the revised manuscript to more comprehensively present the coverage of our imaging system, and we have incorporated this clarification into the Discussion section.

    Method

    (5) More information may be needed about MCNPs:

    a) As reported, there are nanoparticles with different colors in brightfield microscopy, but the particles are also excitable in fluorescence microscopy. Would you please provide a summary about excitation/emission wavelengths of the different MCNPs? This is crucial to understand to what extent the method is compatible with fluorescence immunohistochemistry.

    We thank the reviewer for the careful attention and professional suggestion. We fully agree that this issue is critical for evaluating the compatibility of our method with fluorescent immunohistochemistry. Different types of metal compound nanoparticles (MCNPs) have clearly distinguishable spectral properties:

    - MCNP-Green and MCNP-Yellow: AF488-matched spectra, with excitation/emission wavelengths of 495/519 nm.

    - MCNP-Pink: Designed for far-red spectra, with excitation/emission wavelengths of 561/640 nm.

    - MCNP-Black: Non-fluorescent, appearing black under bright-field microscopy only.

    The above information has been added to the Materials and Methods section.

    b) Also, is there more systematic information available concerning the advantage of these particles compared to 'traditional' fluorescence dyes, such as Alexa fluor or Cy-dyes, in fluorescence microscopy and concerning their compatibility with various tissue clearing methods (e.g., with the frequently used organic-solvent-based methods)?

    We thank the reviewer for the detailed question. Compared with conventional organic fluorescent dyes, MCNP offers the following advantages:

    - Enhanced photostability: Its inorganic core-shell structure resists fading even after hydrogen peroxide bleaching.

    - High signal stability: Fluorescence is maintained during aqueous-based clearing (e.g., CUBIC) and multiple rounds of staining without quenching.

    We appreciate the reviewer’s suggestion. In our Liver-CUBIC system, MCNP nanoparticles exhibited excellent multi-channel labeling stability and fluorescence signal retention. Regarding compatibility with other clearing methods (e.g., SCAFE, SeeDB, CUBIC), since these methods have limited effectiveness for whole-liver clearing (see Figure 2 of Tainaka, et al. 2014) and cannot meet the requirements for high-resolution microstructural imaging in this study, we consider further testing of their compatibility unnecessary.

    In summary, MCNP dye demonstrates superior signal stability and spectral separation compared with conventional organic fluorescent dyes in multi-channel, long-term, high-transparency three-dimensional tissue imaging.

    c) When you perfuse these particles, to which structures do they bind inside the ducts (vessels, bile ducts)? Is the 48h post-fixation enough to keep them inside the tubes/bind them to the vessel walls? Is there any 'wash-out' during the complex cutting/staining procedure? E.g., in Figure 2D: the 'classical' hepatic artery in the portal triad is not visible - but the MCNP apparently penetrated to the adjacent sinusoids at the edge of the lobulus. Also, in Figure 3B, there is a significant mismatch between the MNCP-green (bile duct) signal and the CD19 (epithelium marker) immunostaining. Please discuss these.

    The experimental results showed that following injection, MCNP nanoparticles primarily remained within the vascular and biliary lumens, and their tissue distribution depended on physical perfusion. No dye signal was observed to diffuse into the surrounding parenchyma, nor did the particles adhere to cell surfaces or enter cells. The newly added Supplementary Figures S2A–H further confirm this feature: the dye signal is strictly confined within the lumens, clearly delineating continuous vascular paths and biliary branching patterns, strongly supporting the conclusion that “MCNP dye is distributed only within luminal spaces.”

    Thus, MCNP dye mainly serves as an intraluminal tracer rather than a label for specific cell types.

    We provide the following explanations and analyses regarding MCNP distribution in the hepatic vascular and biliary systems and its post-fixation stability:

    - Potential signal displacement during sectioning/immunostaining: During slicing and immunostaining, a small number of particles may be washed away due to mechanical cutting or washing steps; however, the overall three-dimensional structure retains high spatial fidelity.

    - Observation in Figure 2D: MCNP was seen entering the sinusoidal spaces at the lobule periphery, but hepatic arteries were not visible, likely due to limitations in section thickness. Although arteries were not apparent in this slice, arterial distribution around the portal vein is visible in Figure 2C. It should be noted that Figures 2C, D, and E do not represent whole-liver imaging, so not all regions necessarily contain visible hepatic arteries. For easier identification, the main hepatic artery trunk is highlighted in cyan in Figure 2E.

    - Incomplete biliary signal in Figure 3B: This may be because CK19 labeling only covers biliary epithelial cells, whereas MCNP-green distributes throughout the biliary lumen. In Figure 3B, the terminal MCNP-green signal exhibits irregular polygonal structures, which we interpret as the canalicular regions.

    (6) Which fixative was used for 48h of postfixation (step 6) after MCNP injections?

    After MCNP injection, mouse livers were post-fixed in 4% paraformaldehyde (PFA) for 48 hours. This fixation condition effectively “locks” the MCNP particles within the vascular and biliary lumens, maintaining their spatial positions, while also being compatible with subsequent sectioning and multi-channel immunostaining analyses.

    The above information has been added to the Materials and Methods section

    (7) What is the 'desired thickness' in step 7? In the case of immunostained tissue, a 200-micrometer slice thickness is mentioned. However, based on the Methods, it is not completely clear what the actual thickness of the tissue was that was examined ultimately in the microscopes, and whether or not the clearing preceded the cutting or vice versa.

    We appreciate the reviewer’s question. The “desired thickness” referred to in step 7 of the manuscript corresponds to the thickness of tissue sections used for immunostaining and high-resolution microscopic imaging, which is typically around 200 µm. We selected 200 µm because this thickness is sufficient to observe the PLC structure in its entirety, allows efficient staining, and preserves tissue architecture well. Other researchers may choose different section thicknesses according to their experimental needs.

    In this study, the processing order for immunostained tissue samples was sectioning followed by clearing, as detailed below:

    Section Thickness

    To ensure antibody penetration and preservation of three-dimensional structure, tissue sections were typically cut to ~200 µm. Thicker sections can be used if more complete three-dimensional structures are required, but adjustments may be needed based on antibody penetration and fluorescence detection conditions.

    Clearing Sequence

    After sectioning, slices were processed using the Liver-CUBIC aqueous-based clearing system.

    (8) More information is needed concerning the 'deep-focus microscopy' (Keyence), the applied confocal system, and the THUNDER 'high resolution imaging system': basic technical information, resolutions, objectives (N.A., working distance), lasers/illumination, filters, etc.

    In this study, all liver lobes (left, right, caudate, and quadrate lobes) were subjected to Liver-CUBIC aqueous-based clearing to ensure uniform visualization of MCNP fluorescence and immunolabeling throughout the three-dimensional imaging of the entire liver.

    The above information has been added to the Materials and Methods section.

    Imaging Systems and Settings

    VHX-6000 Extended Depth-of-Field Microscope: Objective: VH-Z100R, 100×–1000×; resolution: 1 µm (typical); illumination: coaxial reflected; transmitted illumination on platform: ON.

    Zeiss Confocal Microscope (980): Objectives: 20× or 40×; image size: 1024 × 1024. Fluorescence detection was set up in three channels:

    - Channel 1: 639 nm laser, excitation 650 nm, emission 673 nm, detection range 673–758 nm, corresponding to Cy5-T1 (red).

    - Channel 2: 561 nm laser, excitation 548 nm, emission 561 nm, detection range 547–637 nm, corresponding to Cy3-T2 (orange).

    - Channel 3: 488 nm laser, excitation 493 nm, emission 517 nm, detection range 490–529 nm, corresponding to AF488-T3 (green).

    Leica THUNDER Imager 3D Tissue: Fluorescence detection in two channels:

    - Channel 1: FITC channel (excitation 488 nm, emission ~520 nm).

    - Channel 2: Orange-red channel (excitation/emission 561/640 nm).
    Equipped with matching filter sets to ensure signal separation.

    The above information has been added to the Materials and Methods section.

    (9) Liver-CUBIC, step 2: which lobe(s) did you clear (...whole liver lobes...).

    In this study, all liver lobes (left, right, caudate, and quadrate lobes) were subjected to Liver-CUBIC aqueous-based clearing to ensure uniform visualization of MCNP fluorescence and immunolabeling throughout the three-dimensional imaging of the entire liver.

    The above information has been added to the Materials and Methods section.

    (10) For the DAB and TSA IHC stainings, did you use free-floating slices, or did you mount the vibratome sections and do the staining on mounted sections?

    In this study, fixed livers were first sectioned into thick slices (~200 µm) using a vibratome. Subsequently, DAB and TSA immunohistochemical (IHC) staining were performed on free-floating sections. During the entire staining process, the slices were kept floating in the solutions, ensuring thorough antibody penetration in the thick sections while preserving the three-dimensional tissue architecture, thereby facilitating multiple rounds of staining and three-dimensional imaging.

    (11) Regarding the 'transmission quantification': this was measured on 1 mm thick slices. While it is interesting to make a comparison between different clearing methods in general, one must note that it is relatively easy to clear 1mm thick tissue slices with almost any kind of clearing technique and in any tissues. The 'real' differences come with thicker blocks, such as >5mm in the thinnest dimension. Do you have such experiences (e.g., comparison in whole 'left lateral liver lobes')?

    In this study, we performed three-dimensional visualization of entire liver lobes to depict the distribution of MCNPs and the overall spatial architecture of the vascular and biliary systems (Figure S1F). However, due to the limitations of the plate reader and fluorescence imaging systems in terms of spatial resolution and light penetration depth, quantitative analyses were conducted only on tissue sections approximately 1 mm thick.

    Regarding the comparative quantification of different clearing methods, as the reviewer noted, nearly all aqueous- or organic solvent–based clearing techniques can achieve relatively uniform transparency in 1 mm-thick tissue sections, so differences at this thickness are limited. We have not yet conducted systematic comparisons on whole-lobe sections thicker than 5 mm and therefore cannot provide “true” difference data for thicker tissues.

    (12) There is no method description for the ELMI studies in the Methods.

    Transmission Electron Microscopy (TEM) Analysis of MCNPs

    Before imaging, the MCNP dye solution was centrifuged at 14,000 × g for 10 minutes at 4 °C to remove aggregates and impurities. The supernatant was collected, diluted 50-fold, and 3–4 μL of the sample was applied onto freshly glow-discharged Quantifoil R1.2/1.3 copper grids (Electron Microscopy Sciences, 300 mesh). The sample was allowed to sit for 30 seconds to enable particle adsorption, after which excess liquid was gently wicked away with filter paper and the grid was air-dried at room temperature. The sample was then negatively stained with 1% uranyl acetate for 30 seconds and air-dried again before imaging.

    Negative-stain TEM images were acquired using a JEOL JEM-1400 transmission electron microscope operating at 120 kV and equipped with a CCD camera. Data acquisition followed standard imaging conditions.

    The above information has been added to the Materials and Methods section.

    (13) Please, provide a method description for the applied CCl4 cirrhosis model. This is completely missing.

    (1) Under a fume hood, carbon tetrachloride (CCl₄) was dissolved in corn oil at a 1:3 volume ratio to prepare a working solution, which was filtered through a 0.2 μm filter into a 30 mL glass vial. In our laboratory, to mimic chronic injury, mice in the experimental group were intraperitoneally injected at a dose of 1 mL/kg body weight per administration.

    (2) Mice were carefully removed from the cage and placed on a scale to record body weight for calculation of the injection volume.

    (3) The needle cap was carefully removed, and the required volume of the pre-prepared CCl₄ solution was drawn into the syringe. The syringe was gently flicked to remove any air bubbles.

    (4) Mice were placed on a textured surface (e.g., wire cage) and restrained. When the mouse was properly positioned, ideally with the head lowered about 30°, the left lower or right lower abdominal quadrant was identified.

    (5) Holding the syringe at a 45° angle, with the bevel facing up, the needle was inserted approximately 4–5 mm into the abdominal wall, and the calculated volume of CCl₄ was injected.

    (6) Mice were returned to their cage and observed for any signs of discomfort.

    (7) Needles and syringes were disposed of in a sharps container without recapping. A new syringe or needle was used for each mouse.

    (8) To establish a progressive liver fibrosis model, injections were administered twice per week (e.g., Monday and Thursday) for 3 or 6 consecutive weeks (n=3 per group). Control mice were injected with an equal volume of corn oil for 3 or 6 weeks (n=3 per group).

    (9) Forty-eight hours after the last injection, mice were euthanized by cervical dislocation, and livers were rapidly harvested. Portions of the liver were processed for paraffin embedding and histological sectioning, while the remaining tissue was either immediately frozen or used for subsequent molecular biology analyses.

    The above information has been added to the Materials and Methods section.

    (14) Please provide a method description for the quantifications reported in Figures 5D, 5F, and 6E.

    ImageJ software was used to analyze 3D stained images (Figs. 5F, 6E), and the ultra-depth-of-field 3D analysis module was used to analyze 3D DAB images (Fig. 5D). The specific steps are as follows:

    Figure 5D: DAB-stained 3D images from the control group and the CCl4 6-week (CCl4-6W) group were analyzed. For each group, 20 terminal bile duct branch nodes were randomly selected, and the actual path distance along the branch to the nearest portal vein surface was measured. All measurements were plotted as scatter plots to reflect the spatial extension of bile ducts relative to the portal vein under different conditions.

    Figure 5F: TSA 3D multiplex-stained images from the control group, CCl4 3-week (CCl4-3W), and CCl4 6-week (CCl4-6W) groups were analyzed. For each group, 5 terminal bile duct branch nodes were randomly selected, and the actual path distance along the branch to the nearest portal vein surface was measured. Measurements were plotted as scatter plots to illustrate bile duct spatial extension.

    Figure 6E: TSA 3D multiplex-stained images from the control, CCl4-3W, and CCl4-6W groups were analyzed. For each group, 5 terminal nerve branch nodes were randomly selected, and the actual path distance along the branch to the nearest portal vein surface was measured. Scatter plots were generated to depict the spatial distribution of nerves under different treatment conditions.

    (15) Please provide a method description for the human liver samples you used in Figure S6. Patient data, fixation, etc...

    The human liver tissue samples shown in Figure S6 were obtained from adjacent non-tumor liver tissues resected during surgical operations at West China Hospital, Sichuan University. All samples used were anonymized archived tissues, which were applied for scientific research in accordance with institutional ethical guidelines and did not involve any identifiable patient information. After being fixed in 10% neutral formalin for 24 hours, the tissues were routinely processed for paraffin embedding (FFPE), and sectioned into 4 μm-thick slices for immunostaining and fluorescence imaging.

    Results

    (16) While it is stated in the Methods that certain color MCNPs were used for labelling different structures (i.e., yellow: hepatic artery; green: bile duct; portal vein: pink; central veins: black), in some figures, apparently different color MCNPs are used for the respective structures. E.g., in Figure 1J, the artery is pink and the portal vein is green. Please clarify this.

    The color assignment of MCNP dyes is not fixed across different experiments or schematic illustrations. MCNP dyes of different colors are fundamentally identical in their physical and chemical properties and do not exhibit specific binding or affinity for particular vascular structures. We select different colors based on experimental design and imaging presentation needs to facilitate distinction and visualization, thereby enhancing recognition in 3D reconstruction and image display. Therefore, the color labeling in Figure 1F is primarily intended to illustrate the distribution of different vascular systems, rather than indicating a fixed correspondence to a specific dye or injection color.

    (17) In Figure 1J, the hepatic artery is extremely shrunk, while the portal vein is extremely dilated - compared to the physiological situation. Does it relate to the perfusion conditions?

    We appreciate the reviewer’s attention. In fact, under normal physiological conditions, the hepatic arteries labeled by CD31 are naturally narrow. Therefore, the relatively thin hepatic arteries and thicker portal veins shown in Figure 1J are normal and unrelated to the perfusion conditions. See figure 1E of Adori et al., 2021.

    (18) Re: MCNP-black labelled 'oval fenestrae': the Results state 50-100 nm, while they are apparently 5-10-micron diameter in Figure 1I. Accordingly, the comparison with the ELMI studies in the subsequent paragraph is inappropriate.

    We thank the reviewer for the correction. The previous statement was a typographical error. In fact, the diameter of the “elliptical windows” marked by MCNP-black is 5–10 μm, so the diameter of 5–10 μm shown in Figure 1I is correct.

    (19) Please, correct this erroneous sentence: 'Pink marked the hepatic arterial system by injection extrahepatic duct (Figure 2B).'

    Original sentence: “The hepatic arterial system was labeled in pink by injection through the extrahepatic duct (Figure 2B).”

    Revised sentence: “The hepatic arterial system was labeled in pink by injection through the left ventricle (Figure 2B).”

    (20) How do you define the 'primary portal vein tract'?

    We thank the reviewer for the question. The term “primary portal vein tract” refers to the first-order branches of the portal vein that enter the liver from the hepatic hilum. These are the major branches arising directly from the main portal vein trunk and are responsible for supplying blood to the respective hepatic lobes. This definition corresponds to the concept of the first-order portal vein in hepatic anatomy.

    (21) I am concerned that the 'periportal lamellar complex (PLC)' that the Authors describe really exists as a distinct anatomical or functional unit. I also see these in 3D scans - in my opinion, these are fine, lower-order portal vein branches that connect the portal veins to the adjacent sinusoid. The strong MCNP-labelling of these structures may be caused by the 'sticking' of the perfused MCNP solutions in these 'pockets' during the perfusion process. What do these structures look like with SMA or CD31 immunostaining? Also, one may consider that the anatomical evaluation of these structures may have limitations in tissue slices. Have you ever checked MCNP-perfused, cleared full live lobes in light sheet microscope scans? I think this would be very useful to have a comprehensive morphological overview. Unfortunately, based on the presented documentation, I am also not convinced that PLCs are 'co-localize' with fine terminal bile duct branches (Figure 3E, S3C), or with TH+ 'neuronal bead chain networks' (Fig 6C). More detailed and more convincing documentation is needed here.

    We thank the reviewer for the detailed comments. Regarding the existence and function of the periportal lamellar complex (PLC), our observations are based on MCNP-Pink labeling of the portal vein, through which we were able to identify the PLC structure surrounding the portal branches. It should be noted that the PLC represents a very small anatomical structure. Although we have not yet performed light-sheet microscopy scanning, we anticipate that such imaging would primarily visualize larger portal vein branches. Nevertheless, this does not affect our overall conclusions.

    We also appreciate the reviewer’s suggestion that the observed structures might result from MCNP adherence during perfusion. To verify the structural characteristics of the PLC, we performed immunostaining for SMA and CD31, which revealed a specific arrangement pattern of smooth muscle and endothelial markers rather than simple perfusion-induced deposition (Figures 4F and S6B).

    Regarding the apparent colocalization of the PLC with terminal bile duct branches (Figures 3E and S3C) and TH⁺ neuronal bead-like networks (Figure 6C), we acknowledge that current literature evidence remains limited. Therefore, we have carefully described these observations as possible spatial associations rather than definitive conclusions. Future studies integrating high-resolution three-dimensional imaging with functional analyses will help to further clarify the anatomical and physiological significance of the PLC.

    (22) 'Extended depth-of-field three-dimensional bright-field imaging revealed a strict 1:1 anatomical association between the primary portal vein trunk (diameter 280 {plus minus} 32 μm) and the first-order bile duct (diameter 69 {plus minus} 8 μm) (Figures 3A and S3A)'.

    How do you define '1:1 anatomical association'? How do you define and identify the 'order' (primary, secondary) of vessel and bile duct branches in 200-micrometer slices?

    We thank the reviewer for the question. In this study, the term “1:1 anatomical correlation” refers to the stable paired spatial relationship between the main portal vein trunk and its corresponding primary bile duct within the same portal territory. In other words, each main portal vein branch is accompanied by a primary bile duct of matching branching order and trajectory, together forming a “vascular–biliary bundle.”

    The definitions of “primary” and “secondary” branches were based on extended-depth 3D bright-field reconstructions, considering both branching hierarchy and vessel/duct diameters: primary branches arise directly from the main trunk at the hepatic hilum and exhibit the largest diameters (averaging 280 ± 32 μm for the portal vein and 69 ± 8 μm for the bile duct), whereas secondary branches extend from the primary branches toward the lobular interior with smaller calibers.

    (23) In my opinion, the applied methodical approach in the single cell transcriptomics part (data mining in the existing liver single cell database and performing Venn diagram intersection analysis in hepatic endothelial subpopulations) is largely inappropriate and thus, all the statements here are purely speculative. In my opinion, to identify the molecular characteristics of such small and spatially highly organized structures like those fine radial portal branches, the only way is to perform high-resolution spatial transcriptomic.

    We thank the reviewer for the comment. We fully acknowledge the importance of high-resolution spatial transcriptomics in identifying the fine structural characteristics of portal vein branches. Due to current funding and technical limitations, we were unable to perform such high-resolution spatial transcriptomic analyses. However, we validated the molecular features of the PLC using another publicly available liver single-cell RNA-sequencing dataset, which provided preliminary supporting evidence (Figures S6B and S6C). In the manuscript, we have carefully stated that this analysis is exploratory in nature and have avoided overinterpretation. In future studies, high-resolution spatial omics approaches will be invaluable for more precisely delineating the molecular characteristics of these fine structures.

    (24) 'How the autonomic nervous system regulates liver function in mice despite the apparent absence of substantive nerve fiber invasion into the parenchyma remains unclear.'

    Please consider the role of gap junctions between hepatocytes (e.g., Miyashita, 1991; Seseke, 1992).

    In this study, we analyzed the spatial distribution of hepatic nerves in mice using immunofluorescence staining and found that nerve fibers were almost exclusively confined to the portal vein region (Figure S6A). Notably, this distribution pattern differs markedly from that in humans. Previous studies have shown that, in human livers, nerves are not only located around the portal veins but also present along the central veins, interlobular septa, and within the parenchymal connective tissue (Miller et al., 2021; Yi, la Fleur, Fliers & Kalsbeek, 2010).

    Further research has provided a physiological explanation for this interspecies difference: even among species with distinct sympathetic innervation patterns in the parenchyma—i.e., with or without direct sympathetic input—the sympathetic efferent regulatory functions may remain comparable (Beckh, Fuchs, Ballé & Jungermann, 1990). This is because signals released from aminergic and peptidergic nerve terminals can be transmitted to hepatocytes through gap junctions as electrical signals (Hertzberg & Gilula, 1979; Jensen, Alpini & Glaser, 2013; Seseke, Gardemann & Jungermann, 1992; Taher, Farr & Adeli, 2017).

    However, the scarcity of nerve fibers within the mouse hepatic parenchyma suggests that the mechanisms by which the autonomic nervous system regulates liver function in mice may differ from those in humans. This observation prompted us to further investigate the potential role of PLC endothelial cells in this process.

    (25) Please, correct typos throughout the text.

    We thank the reviewer for this comment. We have carefully proofread the entire manuscript and corrected all typographical errors and minor language issues throughout the text.

    Reviewer #3 (Recommendations for the authors):

    (1) A strong recommendation - the authors ought to challenge their scRNAsq- re-analysis with another scRNAseq dataset, namely a recently published atlas of adult liver endothelial, but also mesenchymal, immune, and parenchymal cell populations https://pubmed.ncbi.nlm.nih.gov/40954217/, performed with Smart-seq2 approach, which is perfectly suitable as it brings higher resolution data, and extensive cluster identity validation with stainings. Pietilä et al. indicate a clear distinction of portal vein endothelial cells into two populations that express Adgrg6, Jag1 (e2c), from Vegfc double-positive populations (e5c and e2c). Moreover, the dataset also includes the arterial endothelial cells that were shown to be part of the PLC, but were not followed up with the scRNAseq analysis. This distinction could help the authors to further validate their results, better controlling for cross-contaminations that may occur during scRNAseq preparation.

    We thank the reviewer for the valuable suggestion. As noted, we have further validated the molecular characteristics of the PLC using a recently published atlas of adult liver endothelial cells (Pietilä et al., 2023, PMID: 40954217). This dataset, generated using the Smart-seq2 technique, provides high-resolution transcriptomic profiles. By analyzing this dataset, we identified a CD34⁺LY6A⁺ portal vein endothelial cell population within the e2 cluster, which is localized around the portal vein. We then examined pathways and gene expression patterns related to hematopoiesis, bile duct formation, and neural signaling within these cells. The results revealed gene enrichment patterns consistent with those observed in our primary dataset, further supporting the robustness of our analysis of the PLC’s molecular characteristics.

    (2) Improving the methods section is highly recommended, this includes more detailed information for material and protocols used - catalog numbers; protocol details of the usage - rocking platforms, timing, and tubes used for incubations; GitHub or similar page with code used for the scRNA seq re-analysis.

    We thank the reviewer for the valuable suggestion. We have added more detailed information regarding the materials and experimental procedures in the Methods section, including catalog numbers, incubation conditions (such as the type of shaker, incubation time, and tube specifications), and other relevant parameters.

    (3) In Figure 2A, the authors claim the size of the nanoparticle is 100nm, while based on the image, the size is ~150-180nm. A more thorough quantification of the particle size would help users estimate the usability of their method for further applications.

    We thank the reviewer for the comment. In the TEM image shown in Figure 2A, the nanoparticles indeed appear to be approximately 150–200 nm in size. We have re-verified the particle dimensions and will update the corresponding description in the Methods section to allow readers to more accurately assess the applicability of this approach.

    (4) In Figure 3E, it is not clear what is labeled by the pink signal. Please consider labeling the structures in the figure.

    We thank the reviewer for the valuable comment. The pink signal in Figure 3E was originally intended to label the hepatic artery. However, a slight spatial misalignment occurred during the labeling process, making its position appear closer to the central vein rather than the portal vein in the image. To avoid misunderstanding, we will add clear annotations to the image and clarify this deviation in the figure legend in the revised version. It should also be noted that this figure primarily aims to illustrate the spatial relationship between the bile duct and the portal vein, and this minor deviation does not affect the reliability of our experimental conclusions.

    (5) The following statement is not backed by quantification as it ought to be „Dual-channel three-dimensional confocal imaging combined with CK19 immunostaining revealed that the sites of dye leakage did not coincide with the CK19-positive terminal bile duct epithelium, but instead were predominantly localized within regions adjacent to the PLC structures".

    We thank the reviewer for the valuable comment. We have added the corresponding quantitative analysis to support this conclusion. Quantitative assessment of the extended-depth imaging data revealed that dye leakage predominantly occurred in regions adjacent to the PLC structure, rather than in the perivenous sinusoidal areas. The corresponding results have been presented in the revised Figure 3G.

    (6) Similarly, Figure 4F is central to the Sca1CD34 cell type identification but lacks any quantification, providing it would strengthen the key statement of the article. A possible way to approach this is also by FACS sorting the double-positive cells and bluk/qRT validation.

    We thank the reviewer for raising this point. We agree that quantitative validation of the Sca1⁺CD34⁺ population by FACS sorting could further support our conclusions. However, the primary focus of this study is on the spatial localization and transcriptional features of PLC endothelial cells. The identification of the Sca1⁺CD34⁺ subset is robustly supported by multiple complementary approaches, including three-dimensional imaging, co-staining with pan-endothelial markers, and projection mapping analyses. Collectively, these lines of evidence provide a solid basis for characterizing this unique endothelial population.

    (7) The images in Figure S4D are not comparable, as the Sca1-stained image shows a longitudinal section of the PV, but the other stainings are cross-sections of PVs.

    We thank the reviewer for the careful comment. We agree that the original Sca1-stained image, being a longitudinal section of the portal vein, was not optimal for direct comparison with other cross-sectional images. We have replaced it with a cross-sectional image of the portal vein to ensure comparability across all images. The updated image has been included in the revised Supplementary Figure S4D.

    (8) I might be wrong, but Figure 4J is entirely missing, and only a cartoon is provided. Either remove the results part or provide the data.

    We appreciate the reviewer’s careful observation. Figure 4J was intentionally designed as a schematic illustration to summarize the structural relationships and spatial organization of the portal vein, hepatic artery, and PLC identified in the previous panels (Figures 4A–4I). It does not represent newly acquired experimental data, but rather serves to provide a conceptual overview of the findings.

    To avoid misunderstanding, we have clarified this point in the figure legend and the main text, stating that Figure 4J is a schematic summary rather than an experimental image. Therefore, we respectfully prefer to retain the schematic figure to aid readers’ interpretation of the preceding results.

    (9) The methods section lacks information about the CCL4concentration, and it is thus hard to estimate the dosage of CCL4 received (ml/kg). This is important for the interpretation of the severity of the fibrosis and presence of cirrhosis, as different doses may or may not lead to cirrhosis within the short regimen performed by the authors [PMID: 16015684 DOI: 10.3748/wjg.v11.i27.4167]. Validation of the fibrosis/cirrhosis severity is, in this case, crucial for the correct interpretation of the results. If the level of cirrhosis is not confirmed, only progressive fibrosis should be mentioned in the manuscript, as these two terms cannot be used interchangeably.

    Thank you for the reviewer’s comment. We indeed omitted the information on the concentration of carbon tetrachloride (CCl4) in the Methods section. In our experiments, mice received intraperitoneal injections of CCl4 at a dose of 1 mL/kg body weight, twice per week, for a total of six weeks. We have revised the manuscript accordingly, using the term “progressive fibrosis” to avoid confusion between fibrosis and cirrhosis.

    (10) The following statement is not backed by any correlation analysis: "Particularly during liver fibrosis progression, the PLC exhibits dynamic structural extension correlating with fibrosis severity,.. ".

    We thank the reviewer for the comment. The original statement that the “PLC correlates with fibrosis severity” lacked support from quantitative analysis. To ensure a precise description, we have revised the sentence as follows: “During liver fibrosis progression, the PLC exhibits dynamic structural extension.”

    (11) Similarly, the following statement is not followed by data that would address the impact of innervation on liver function: "How the autonomic nervous system regulates liver function in mice despite the apparent absence of substantive nerve fiber invasion into the parenchyma remains unclear.".

    This section has been revised. In this study, we analyzed the spatial distribution of nerves in the mouse liver using immunofluorescence staining. The results showed that nerve fibers were almost entirely confined to the portal vein region (Figure S6A). Notably, this distribution pattern differs significantly from that in humans. Previous studies have demonstrated that in the human liver, nerves are not only distributed around the portal vein but also present in the central vein, interlobular septa, and connective tissue of the hepatic parenchyma (Miller et al., 2021; Yi, la Fleur, Fliers & Kalsbeek, 2010).

    Previous studies have further explained the physiological basis for this difference: even among species with differences in parenchymal sympathetic innervation (i.e., species with or without direct sympathetic input), their sympathetic efferent regulatory functions may still be similar (Beckh, Fuchs, Ballé & Jungermann, 1990). This is because signals released by adrenergic and peptidergic nerve terminals can be transmitted to hepatocytes as electrical signals through intercellular gap junctions (Hertzberg & Gilula, 1979; Jensen, Alpini & Glaser, 2013; Seseke, Gardemann & Jungermann, 1992; Taher, Farr & Adeli, 2017). However, the scarcity of nerve fibers in the mouse hepatic parenchyma suggests that the mechanism by which the autonomic nervous system regulates liver function in mice may differ from that in humans. This finding also prompts us to further explore the potential role of PLC endothelial cells in this process.

    (12) Could the authors discuss their interpretation of the results in light of the fact that the innervation is lower in cirrhotic patients? https://pmc.ncbi.nlm.nih.gov/articles/PMC2871629/. Also, while ADGRG6 (Gpr126) may play important roles in liver Schwann cells, it is likely not through affecting myelination of the nerves, as the liver nerves are not myelinated https://pubmed.ncbi.nlm.nih.gov/2407769/ and https://www.pnas.org/doi/10.1073/pnas.93.23.13280.

    We have revised the text to state that although most hepatic nerves are unmyelinated, GPR126 (ADGRG6) may regulate hepatic nerve distribution via non-myelination-dependent mechanisms. Studies have shown that GPR126 exerts both Schwann cell–dependent and –independent functions during peripheral nerve repair, influencing axon guidance, mechanosensation, and ECM remodeling (Mogha et al., 2016; Monk et al., 2011; Paavola et al., 2014).

    (13) The manuscript would benefit from text curation that would:

    a) Unify the language describing the PLC, so it is clear that (if) it represents protrusions of the portal veins.

    We have standardized the description of the PLC throughout the manuscript, clearly specifying its anatomical relationship with the portal vein. Wherever appropriate, we indicate that the PLC represents protrusions associated with the portal vein, avoiding ambiguous or inconsistent statements.

    b) Increase the accuracy of the statements.

    Examples: "bile ducts, and the central vein in adult mouse livers."

    We have refined all statements for accuracy.

    c) Reduce the space given to discussion and results in the introduction, moving them to the respective parts. The same applies to the results section, where discussion occurs at more places than in the Discussion part itself.

    We have edited the Introduction, removing detailed results and functional explanations, and retaining only a concise overview.

    Examples: "The formation of PLC structures in the adventitial layer may participate in local blood flow regulation, maintenance of microenvironmental homeostasis, and vascular-stem cell interactions."

    "This finding suggests that PLC endothelial cells not only regulate the periportal microcirculatory blood flow, but also establish a specialized microenvironment that supports periportal hematopoietic regulation, contributing to stem cell recruitment, vascular homeostasis, and tissue repair. "

    "Together, these findings suggest the PLC endothelium may act as a key regulator of bile duct branching and fibrotic microenvironment remodeling in liver cirrhosis. " This one in particular would require further validation with protein stainings and similar, directly in your model.

    d) Provide a clear reference for the used scRNA seq so it's clear that the data were re-analyzed.

    Example: "single-cell transcriptomic analysis revealed significant upregulation of bile duct-related genes in the CD34+Sca-1+ endothelium of PLC in cirrhotic liver, with notably high expression of Lgals1 (Galectin-1) and HGF(Figure 5G) "

    When describing the transcriptional analysis of PLC endothelial cells, we explicitly cited the original scRNA-seq dataset (Su et al., 2021), clarifying that these data were reanalyzed rather than newly generated.

    e) Introducing references for claims that, in places, are crucial for further interpretation of experiments.

    Examples: "It not only guides bile duct branching during development but also"; the authors show no data from liver development.

    Thank you for pointing this out. We have revised the relevant statement to ensure that the claim is accurate and well-supported.

    f) Results sentence "Instead, bile duct epithelial cells at the terminal ducts extended partially along the canalicular network without directly participating in the formation of the bile duct lumen." Lacks a callout to the respective Figure.

    We would like to thank the reviewers for pointing out this issue. In the revised manuscript, the relevant image (Figure 3D) has been clearly annotated with white arrows to indicate the phenomenon of terminal cholangiocytes extending along the bile canaliculi network. Additionally, the schematic diagram on the right side clearly shows the bile canaliculi, cholangiocytes, and bile flow direction using arrows and color coding, thus intuitively corresponding to the textual description.

    (14) Formal text suggestions: The manuscript text contains a lot of missed or excessive spaces and several typos that ought to be fixed. A few examples follow:

    a) "densely n organized vascular network "

    b) "analysis, while offering high spatial "

    c) "specific differences, In the human liver, "

    d) Figure 4F has a typo in the description.

    e) "generation of high signal-to-noise ratio, multi-target " SNR abbreviation was introduced earlier.

    f) Canals of Hering, CoH abbreviation comes much later than the first mention of the Canals of Hering.

    We thank the reviewer for the helpful comment regarding textual consistency. We have carefully reviewed and revised the entire manuscript to improve the accuracy, clarity, and consistency of the text.

  17. eLife

    eLife Assessment

    This study identifies the Periportal Lamellar Complex (PLC), an important new structure revealed by a novel 3D imaging method. However, the evidence supporting its distinct cellular identity and functional role is currently incomplete, as it relies on transcriptomic re-analysis and correlation without direct experimental validation. Addressing the key issues of methodological rigor and providing functional evidence is essential to fully substantiate these significant claims.

  18. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Weaknesses:

    This manuscript is well-written, organized, and informative. However, there are some points that need to be clarified.

    (1) After MCNP-dye injection, does it remain in the blood vessels, adsorb onto the cell surface, or permeate into the cells? Does the MCNP-dye have cell selectivity?

    (2) All MCNP-dyes were injected after the mice were sacrificed, and the mice's livers were fixed with PFA. After the blood flow had ceased, how did the authors ensure that the MCNP-dyes were fully and uniformly perfused into the microcirculation of the liver?

    (3) It is advisable to present additional 3D perspective views in the article, as the current images exhibit very weak 3D effects. Furthermore, it would be better to supplement with some videos to demonstrate the 3D effects of the stained blood vessels.

    (4) In Figure 1-I, the authors used MCNP-Black to stain the central veins; however, in addition to black, there are also yellow and red stains in the image. The authors need to explain what these stains are in the legend.

    (5) There is a typo in the title of Figure 4F; it should be "stem cell".

    (6) Nuclear staining is necessary in immunofluorescence staining, especially for Figure 5e. This will help readers distinguish whether the green color in the image corresponds to cells or dye deposits.

  19. eLife

    Reviewer #2 (Public review):

    Summary:

    The present manuscript of Xu et al. reports a novel clearing and imaging method focusing on the liver. The authors simultaneously visualized the portal vein, hepatic artery, central vein, and bile duct systems by injecting metal compound nanoparticles (MCNPs) with different colors into the portal vein, heart left ventricle, inferior vena cava, and the extrahepatic bile duct, respectively. The method involves: trans-cardiac perfusion with 4% PFA, the injection of MCNPs with different colors, clearing with the modified CUBIC method, cutting 200 micrometer thick slices by vibratome, and then microscopic imaging. The authors also perform various immunostaining (DAB or TSA signal amplification methods) on the tissue slices from MCNP-perfused tissue blocks. With the application of this methodical approach, the authors report dense and very fine vascular branches along the portal vein. The authors name them as 'periportal lamellar complex (PLC)' and report that PLC fine branches are directly connected to the sinusoids. The authors also claim that these structures co-localize with terminal bile duct branches and sympathetic nerve fibers, and contain endothelial cells with a distinct gene expression profile. Finally, the authors claim that PLC-s proliferate in liver fibrosis (CCl4 model) and act as a scaffold for proliferating bile ducts in ductular reaction and for ectopic parenchymal sympathetic nerve sprouting.

    Strengths:

    The simultaneous visualization of different hepatic vascular compartments and their combination with immunostaining is a potentially interesting novel methodological approach.

    Weaknesses:

    This reviewer has several concerns about the validity of the microscopic/morphological findings as well as the transcriptomics results. In this reviewer's opinion, the introduction contains overstatements regarding the potential of the method, there are severe caveats in the method descriptions, and several parts of the Results are not fully supported by the documentation. Thus, the conclusions of the paper may be critically viewed in their present form and may need reconsideration by the authors.

  20. eLife

    Reviewer #3 (Public review):

    Summary:

    In the reviewed manuscript, researchers aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the portal vein axis. The PLC originates from the portal vein and is characterized by a unique population of CD34⁺Sca-1⁺ dual-positive endothelial cells. Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting the mRNA presence of genes linked to neurodevelopment, biliary function, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

    The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver at the same time. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists; however, some claims need more thorough assessment by functional experimental approaches to decipher the functional molecules and the sequence of events before establishing the PLC as the key hub governing the activity of biliary, arterial, and neuronal liver systems. Similarly, the level of detail of the methods section does not appear to be sufficient to exactly recapitulate the performed experiments, which is of concern, given that the new technique is a cornerstone of the manuscript.

    Nevertheless, the work does bring a clear new insight into the liver structure and functional units and greatly improves the methodological toolbox to study it even further, and thus fully deserves the attention of readers.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.

    This work proposes a new biological framework between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - the Periportal Lamellar Complexes (PLCs).

    Weaknesses:

    Possible overinterpretation of the CD34+Sca1+ findings was built on re-analysis of one scRNAseq dataset.

    Lack of detail in the materials and methods section greatly limits the usefulness of the new technique to other researchers.

  21. Version published to 10.7554/elife.108669.1 on eLife
  22. Version published to 10.1101/2025.08.29.673025 on bioRxiv