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ABSTRACT Severe damage to the intrahepatic biliary duct (IHBD) network occurs in multiple human advanced cholangiopathies, such as primary sclerosing cholangitis, biliary atresia and

end-stage primary biliary cholangitis. Whether and how a severely damaged IHBD network could reconstruct has remained unclear. Here we show that, although the gallbladder is not directly

connected to the IHBD, there is a common hepatic duct (CHD) in between, and severe damage to the IHBD network induces migration of gallbladder smooth muscle cells (SMCs) to coat the CHD in

mouse and zebrafish models. These gallbladder-derived, CHD-coating SMCs produce retinoic acid to activate Sox9b in the CHD, which drives proliferation and ingrowth of CHD cells into the

inner liver to reconstruct the IHBD network. This study reveals a hitherto unappreciated function of the gallbladder in the recovery of injured liver, and characterizes mechanisms involved

in how the gallbladder and liver communicate through inter-organ cell migration to drive tissue regeneration. Carrying out cholecystectomy will thus cause previously unexpected impairments

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BEING VIEWED BY OTHERS MULTIDIMENSIONAL IMAGING OF LIVER INJURY REPAIR IN MICE REVEALS FUNDAMENTAL ROLE OF THE DUCTULAR REACTION Article Open access 05 June 2020 BILIARY NIK PROMOTES

DUCTULAR REACTION AND LIVER INJURY AND FIBROSIS IN MICE Article Open access 30 August 2022 IMPACT OF GALLBLADDER HYPOPLASIA ON HILAR HEPATIC DUCTS IN BILIARY ATRESIA Article Open access 11

June 2024 DATA AVAILABILITY All zebrafish lines and plasmids generated in this study will be made available on reasonable request, but we may require a payment or a completed material

transfer agreement if there is potential for commercial application. Data supporting the findings of this study are available from the corresponding author on reasonable request. Source data

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_Hepatology_ 70, 2092–2106 (2019). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank M. Parsons and S. Childs for plasmids, J. Peng for antibodies, C. Xu and B. Zhou

for transgenic mice. This work was supported by the National Natural Science Foundation of China (grants 32430032 (L. Luo), 32192400 (L. Luo), 32122033 (J.H.), 32470881 (J.H.) and 31970784

(J.H.)), the National Key R&D Program of China (grant 2021YFA0805000, L. Luo), the Natural Science Foundation of Chongqing (grant CSTB2023NSCQ-JQX0004, J.H.) and funds from Southwest

University (grants SWU-XJLJ202302 and SWU-XDPY22008, J.H.). AUTHOR INFORMATION Author notes * These authors contributed equally: Jianbo He, Shuang Li. AUTHORS AND AFFILIATIONS * State Key

laboratory of Genetic Engineering, School of Life Sciences, Liver Cancer Institute of Zhongshan Hospital, Fudan University, Shanghai, China Jianbo He, Jianlong Ma, Jingying Chen, Yunfan Sun,

 Tianyu Zhao & Lingfei Luo * Institute of Developmental Biology and Regenerative Medicine, Southwest University, Chongqing, China Jianbo He, Shuang Li, Zhuolin Yang, Chuanfang Qian, 

Zhuofu Huang, Linke Li, Yun Yang & Lingfei Luo Authors * Jianbo He View author publications You can also search for this author inPubMed Google Scholar * Shuang Li View author

publications You can also search for this author inPubMed Google Scholar * Zhuolin Yang View author publications You can also search for this author inPubMed Google Scholar * Jianlong Ma

View author publications You can also search for this author inPubMed Google Scholar * Chuanfang Qian View author publications You can also search for this author inPubMed Google Scholar *

Zhuofu Huang View author publications You can also search for this author inPubMed Google Scholar * Linke Li View author publications You can also search for this author inPubMed Google

Scholar * Yun Yang View author publications You can also search for this author inPubMed Google Scholar * Jingying Chen View author publications You can also search for this author inPubMed 

Google Scholar * Yunfan Sun View author publications You can also search for this author inPubMed Google Scholar * Tianyu Zhao View author publications You can also search for this author

inPubMed Google Scholar * Lingfei Luo View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L. Luo, J.H., J.M., Y.S. and J.C. designed the

experimental strategy, analysed data and wrote the manuscript. S.L. performed and J.M. and T.Z. helped perform all the mice experiments. Z.Y. performed FISH. C.Q. performed zebrafish lineage

tracing. Z.H. and L. Li crossed and identified mice. Y.Y. helped with analyses. J.H. performed all the other experiments. CORRESPONDING AUTHOR Correspondence to Lingfei Luo. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Cell Biology_ thanks Wolfram Goessling and the other, anonymous,

reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published

maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 THE RECONSTRUCTION OF SEVERELY DAMAGED IHBD NETWORK AND INVOLVEMENTS OF GALLBLADDER IN THE ANIT-INJURED MOUSE. A, IHBD

system visualized by retrograde ink injection into CBD of α-naphthylisothiocyanate (ANIT)-treated mice at 3 dpi (n = 7/10), 7 dpi (n = 7/11), 12 dpi (n = 8/10), and 28 dpi (n = 8/11). The

dashed lines indicate the gallbladder. B, IHBD system visualized by retrograde ink injection into CBD of ANIT-treated mice at 12 dpi in cholecystectomy group (n = 6/8). C, IHBD system

visualized by retrograde ink injection into CBD of ANIT-treated mice at 12 dpi in BMS493-treated group (n = 5/7). The dashed lines indicate the gallbladder. Scale bars, 200 μm. EXTENDED DATA

FIG. 2 THE GALLBLADDER IS ESSENTIAL FOR IHBD NETWORK RECONSTRUCTION AFTER SEVERE IHBD DAMAGES IN ADULT ZEBRAFISH. A, Experimental strategies of IHBD injury and reconstruction in adult

zebrafish. B, Confocal projection images for Tomato+ IHBD network reconstruction of uninjured group (n = 5/5) and MTZ-treated groups at 2 dpi (n = 8/8), 6 dpi (n = 9/12), 21 dpi (n = 12/15),

56 dpi (n = 11/14), and 84 dpi (n = 8/10). The dashed lines indicate the migration of IHBD cells and the asterisk indicates the pancreatic region outside of the liver. C, Diagram indicating

the IHBD network reconstruction starts from the region near gallbladder, fulfills in the left lobe and extends to the middle lobe, further extends to the right lobe, and completely

accomplishes in the whole liver. D, Experimental strategies of cholecystectomy. The undistinguished gallbladders (n = 10/10) labeled with methyl blue (n = 8/8) and subjected to

cholecystectomy (n = 12/12). E, Confocal projection images for Tomato+ IHBD reconstruction from sham-treated (n = 8/11) and cholecystectomy-treated (n = 11/12) zebrafish at 10 dpi. The

dashed lines indicate new regenerating IHBD cells. Scale bars, 500 μm. EXTENDED DATA FIG. 3 CHOLECYSTECTOMY IN ZEBRAFISH. A, Graphic images illustrate the procedures of cholecystectomy in

zebrafish adult. B, Graphic images illustrate the procedures of cholecystectomy in zebrafish larva. C, Extrahepatic duct system including CHD, cystic duct (CD), and GB visualized under the

_Tg(anxa4:GFP)_ transgenic background after Sham (n = 5/5) or cholecystectomy (n = 7/7) in adult zebrafish. Note that cholecystectomy removes GB and CD, but leaves CHD intact. D,

Extrahepatic duct system including CHD and GB visualized under the _Tg(anxa4:GFP)_ transgenic background at 5 dpf/0 dpc, 6 dpf/1 dpc, and 10 dpf/5 dpc after Sham (n = 10/10) or

cholecystectomy (n = 10/10) in larval zebrafish. The larvae were subjected to cholecystectomy at 5 dpf. Note that cholecystectomy removes GB, but leaves CHD intact. dpc, days post

cholecystectomy. Scale bars, 2 mm (C); 100 μm (D). The models (A, B) were Created in BioRender. EXTENDED DATA FIG. 4 THE MTZ INDUCES SEVERE AND SPECIFIC INJURY TO IHBDS IN ZEBRAFISH LARVAE.

A, Experimental strategies of MTZ treatment and analyses. B, The confocal projection images and quantifications for the Tomato+ IHBD cells at 1 dpi in the larvae subjected to treatments of

DMSO (n = 5 larvae), 5 mM MTZ (n = 12 larvae), 8 mM MTZ (n = 13 larvae), and 10 mM MTZ (n = 7 larvae). The dashed lines indicate the liver. C, Confocal images and quantifications for the

TUNEL assays at 0 dpi after DMSO (n = 6 larvae) or MTZ (n = 6 larvae) treatment. Note that the TUNEL+ cells are exclusively present in the inter-hepatocyte space, which means apoptotic BECs.

The anti-Bhmt indicate hepatocytes. D, The confocal images of anti-Anxa4, GFP, and Tomato in the DMSO- and MTZ-treated groups at 1 dpi. The dashed lines indicate the liver. Quantifications

indicate the number of Anxa4+Tomato+ BECs in the liver at 1 dpi after DMSO (n = 6 larvae) or MTZ (n = 5 larvae) treatment. E, The confocal projection images for anti-SPGP at 1 dpi after DMSO

(n = 10/10) and MTZ (n = 7/11) treatment. The dashed lines indicate the liver. F, The confocal projection images for anti-Anxa4 at 1 dpi. The dashed lines indicate the gallbladder.

Quantifications indicate the areas of gallbladder at 1 dpi after DMSO (n = 6 larvae) or MTZ (n = 10 larvae) treatment. G, The bright field (BF) and fluorescent images of larval morphologies

and Tomato expression at 1 dpi after DMSO (n = 15/15) or 10 mM MTZ (n = 20/20) treatment. The arrows indicate the liver region. Data are mean±s.e.m.; unpaired _t_-test. Scale bars, 100 μm

(B, D, E, F, G); 50 μm (C). Source data EXTENDED DATA FIG. 5 THE LABELING EFFICIENCY OF CHD CELLS BY THE CRE/LOXP SYSTEM IS AVERAGELY 88% IN ZEBRAFISH LARVAE. A, Experimental strategies of

CHD labeling with _Cre/loxP_ system. B, Antibody staining confocal images for Anxa4 and GFP in DMSO- and 4-OHT-treated groups at 7 dpf. Quantification of the ratio of GFP+ cells among CHD

after DMSO (n = 5 zebrafish) or 4-OHT (n = 6 zebrafish) treatment at 7 dpf. Data are mean±s.e.m.; unpaired _t_-test. Scale bars, 100 μm. Source data EXTENDED DATA FIG. 6 THE LABELING

EFFICIENCY OF CHD CELLS BY THE CRE/LOX-DRE/ROX SYSTEM IS AVERAGELY 80% IN MOUSE. A, Experimental strategies of CHD labeling in Krt19: DreER x Alb: Cre x DeaLT-IR triple transgenic mice after

7 times of 4-OHT intraperitoneal injection and analysis at 20 dpi. B, Diagram showing the section region of CHD (Red dashed line). C, tdTomato, CK19, and ZsGreen immunofluorescent confocal

images of CHD in oil-treated and 4-OHT-treated mice at 20 dpi. D, Quantification of the ratio of tdTomato+ among CK19+ CHD cells at 7 dpi after oil (n = 5 mice) and 4-OHT (n = 5 mice)

treatment. Data are mean±s.e.m.; unpaired _t_-test. Scale bars, 50 μm. Source data EXTENDED DATA FIG. 7 THE _RARGA_ IS TRANSCRIPTIONALLY ACTIVATED IN THE CHD AFTER IHBD INJURIES IN

ZEBRAFISH. FISH combined with anti-Anxa4 antibody staining showing the expressions of _raraa_ (A; DMSO, n = 14/14; MTZ, n = 15/15), _rarab_ (B; DMSO, n = 15/15; MTZ, n = 14/14), _rarga_ (C;

DMSO, n = 16/16; MTZ, n = 10/16), and _rargb_ (D; DMSO, n = 15/15; MTZ, n = 16/16) in the CHD at 1 dpi after MTZ treatment. Note that only _rarga_ was transcriptionally activated in CHD

after MTZ treatment. The dashed lines indicate the CHD region. Scale bars, 100 μm. EXTENDED DATA FIG. 8 RA DRIVES CHD CELL PROLIFERATION AND INGROWTH THROUGH SOX9B IN ZEBRAFISH. A,

Experimental strategies of RA signaling inhibition plus IHBD injury and analysis in zebrafish larvae. FISH combined with antibody staining images for _sox9b_ expressions in Anxa4+ CHD after

MTZ treatment with _hsp70l_- (n = 12/13), _cyp26a1_+ (n = 7/10), and _DnRAR_+ (n = 12/16) overexpression and DMSO-treated control (n = 8/8) groups at 1 dpi in zebrafish. The dashed lines

indicate the CHD. B, Antibody staining confocal images for Anxa4 and PCNA in _sox9b_ sibling and mutant CHD at 1 dpi. Quantification of the ratio of PCNA+ among CHD cells in _sox9b_ sibling

(n = 6 larvae) and mutant (n = 5 larvae) at 1 dpi. The dashed lines indicate the CHD. C, Confocal projection images for new regenerating Tomato+ IHBD cells in _sox9b_ sibling (n = 36/50) and

mutant (n = 40/59) at 20 dpi. Quantification of the ratio of IHBD regenerated larvae in _sox9b_ sibling (n = 3 groups) and mutant (n = 3 groups) at 20 dpi. The dashed line indicates the

IHBD network. Data are mean±s.e.m.; unpaired _t_-test. Scale bars, 100 μm. Source data EXTENDED DATA FIG. 9 THE PROLIFERATION OF IHBD CELL OCCURS AFTER THE MDA-INDUCED INJURY. A, EpCAM and

PCNA immunofluorescent confocal images of IHBD cells in uninjured, MDA-treated, and MDA plus BMS493-treated groups at 6 dpi. Note that the proliferation of IHBD was partially repressed after

BMS493 treatment at 6 dpi. B, The quantification of the ratios of PCNA+ cells among all the EpCAM+ IHBD cells in the uninjured (n = 5 mice), MDA-treated (n = 5 mice), and MDA plus

BMS493-treated (n = 5 mice) groups at 6 dpi. Data are mean±s.e.m.; unpaired _t_-test. Scale bars, 25 μm. Source data SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY TABLE 1

Antibodies and primers used in this study. SUPPLEMENTARY VIDEO 1 Adult zebrafish cholecystectomy. SUPPLEMENTARY VIDEO 2 Larval zebrafish cholecystectomy. SOURCE DATA SOURCE DATA FIG. 1

Statistical source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA

FIG./TABLE 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG./TABLE 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG./TABLE 6 Statistical source data. SOURCE DATA EXTENDED

DATA FIG./TABLE 8 Statistical source data. SOURCE DATA EXTENDED DATA FIG./TABLE 9 Statistical source data. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other

partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this

article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE He, J., Li, S., Yang, Z. _et al._

Gallbladder-derived retinoic acid signalling drives reconstruction of the damaged intrahepatic biliary ducts. _Nat Cell Biol_ 27, 39–47 (2025). https://doi.org/10.1038/s41556-024-01568-8

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