Regional mechanisms governing developmental and regenerative morphology of intrahepatic bile ducts Restricted; Files Only
Hrncir, Hannah (Spring 2025)
Abstract
Intrahepatic bile ducts (IHBDs) form a complex, hierarchical network, with a large bile duct entering at the liver hilum and progressively branching into smaller ducts that terminate as ductules. IHBDs are critical for liver function and chronic loss of IHBD function will lead to liver failure. However, the processes governing their morphogenesis beyond initial specification remain largely undefined. While IHBD remodeling is a key feature of both development and liver disease, the mechanisms regulating this process are not well understood. IHBD remodeling following injury is shaped by the response of biliary epithelial cells to stress, which we characterize by key hallmarks: cell death, proliferation, transdifferentiation, senescence, and the acquisition of a neuroendocrine phenotype. Due to limitations in imaging and analysis, global 3D IHBD remodeling during development and disease remains largely unexplored. To address this gap, we employ large-volume light sheet microscopy and quantitative analysis to characterize IHBD architecture across development, homeostasis, and injury. We demonstrate that the transcription factor Sox9 is required for postnatal branching morphogenesis of ductules by repressing Activin A. This identifies a previously unrecognized postnatal regulatory mechanism distinguishing duct and ductule formation. Additionally, we establish a 3D imaging and computational pipeline to quantitatively assess IHBD structure in both mouse and human liver tissues. Using this approach in models of cholestasis and hepatocellular injury, we identify region-specific IHBD remodeling, including the formation of IHBD diverticula on large ducts following cholestasis. Using high resolution 3D imaging, we visualize the Canal of Herring and its frequent localization with hybrid hepatocytes. This work establishes a mechanistic framework for understanding IHBD hierarchy and remodeling. By examining IHBD remodeling in health and disease, we identify potential therapeutic targets, including Activin A, for cholangiopathies and liver regeneration.
Table of Contents
- CHAPTER 1 – Introduction………………………………………….…………………………………1
1.1 Functional compartmentalization of the liver…………………………………..………...……2
1.2 Formation and differentiation of liver epithelium…………………………………...…………3
1.3 Tissue remodeling in response to liver injury……………………………………………….…5
1.4 Previous work studying 3D organization of intrahepatic bile ducts……………………...……6
1.5 Scope of the dissertation……………………………………………………………………….7
1.6 Figures………………………………………………………………………….……………..10
Figure 1. Liver functions are compartmentalized………………………….…………….10
Figure 2. Intrahepatic bile duct develop through specification and remodeling…..……..11
1.7 References…………………………………………...……………………………….……….12
- CHAPTER 2 – Panic at the bile duct: how intrahepatic cholangiocytes respond to stress and injury……………………………………………………………………………...………………………17
2.1 Abstract………..……………………………………………………...………………………18
2.2 Ductular reaction: a common, but heterogeneous response to liver injury………………..…19
2.3 Cholangiopathies………………………………………………………….…………………..22
2.4 Common BEC responses to injury……………………………………………………………26
2.5 Integrating individual features of BEC stress response to understand BEC pathophysiology.39
2.6 Conclusion………………………………………………………...………………………….41
2.7 Acknowledgements…………………………………………………….….………………….43
2.8 Author contributions…………………………………………………………..……………...43
2.9 Funding sources……………………………………………………………..…………..……43
2.10 Figures……………………………………………………………………..….......................44
Figure 1: Ductular reaction can be classified by parenchymal invasiveness…………….44
Figure 2: Common cellular responses in DR and cholangiopathies……………………..45
Figure 3: Biomarkers and pathways associated with biliary epithelial cell responses to stress and injury…………………………………………..……………………………...46
2.11 References……………………………….…………………………………………….…….47
- CHAPTER 3 – Sox9 inhibits Activin A to promote biliary maturation and branching morphogenesis……………………………………………………………………………………………58
3.1 Abstract……………………………………………………..……………………………...…59
3.2 Introduction…………………………………………………………..……..………………...60
3.3 Results………………………………………….……………………………….…………….61
Ductal paucity in adult Sox9cKO mice……………………………………………….…61
Sox9 is required for proper IHBD morphology……………………………………….…63
Sox9cKO BECs exhibit elevated TGF-β signaling and transcriptomic features of immaturity………………………………...……………………………………..……….64
Sox9 has subpopulation-specific impacts on BEC gene expression…………………..…66
Sox9 is required for normal biliary organoid formation and cystic morphology……..….67
Activin A signaling promotes non-cystic mICO phenotype……………………………..68
Late IHBD development proceeds via proliferative expansion of a continuous ductal “web”……………...……………………………………………………………………..70
Loss of Sox9 results in early postnatal ductal paucity…………………………………...71
Activin A inhibition partially rescues branching morphogenesis in Sox9cKO livers……………………………………………………………………………….……..73
3.4 Discussion……………………………...……………………………………………………..74
3.5 Methods……………………………………………………..………………….……………..80
Animal studies………………………………………………………………...…………80
Perfusion and tissue processing………………………………………………………….80
Intrahepatic bile duct dissociation and FACS……………………………………..……..81
Mouse intrahepatic biliary organoid culture……………………………….…………….83
Immunofluorescence on tissue sections………………………………...………………..85
Whole mount organoid staining………………………………………………………….87
Whole tissue staining and iDISCO+ clearing…………………………………………....89
Light sheet imaging………………………………………………………………………91
Whole tissue image processing and data analysis………………………………………..92
Bulk RNA-sequencing…………………………………...…………………………..…..93
Single cell RNA-sequencing………………………………………………...…………...94
RT-qPCR…………………………………………………………………………………97
3.6 Data availability……………………………………………...……………………………….97
3.7 Acknowledgements……………………………………………..…………………………….98
3.8 Author contributions statement…………………………………………………………….…99
3.9 Competing interests statement…………………………………………………………….….99
3.10 Figures……..………………………………………………….……………………………100
Fig. 1: Loss of Sox9 during development results in ductal paucity in adult mice…..….100
Fig. 2: Sox9 promotes biliary identity and limits TGF-β signaling in BECs……….….102
Fig. 3: Sox9 has subpopulation-specific impacts on BEC gene expression………….…104
Fig. 4: Sox9 promotes cystic morphology in BEC-derived organoids………………….106
Fig. 5: Inhibiting Activin A signaling rescues biliary organoid morphology…………..107
Fig. 6: Whole tissue imaging of EpCAM+ IHBD development………………………..109
Fig. 7: Sox9 regulates branching morphogenesis during IHBD development………….111
Fig. 8: Activin A inhibits IHBD branching morphogenesis during postnatal development…………………………………………………………………………….113
Supplementary Fig. 1: Efficient Sox9 recombination in BECs…………………..……..115
Supplementary Fig. 2: Sox9 promotes IHBD cilia formation but is dispensable for apical polarity and proliferation…………………………………………………………….…117
Supplementary Fig. 3: Sox9cKO IHBDs exhibit regional ductal paucity………..…….119
Supplementary Fig. 4: Transcriptomic analysis of BECs in control and Sox9cKO mice…………………………………….……………………………………………….121
Supplementary Fig. 5: Sox9 regulates gene expression in ducts and ductules………....123
Supplementary Fig. 6: Distinct gene expression in ducts and ductules………...………125
Supplementary Fig. 7: Loss of Sox9 does not impact biliary organoid proliferation…..126
Supplementary Fig. 8: Sox9 promotes IHBD branching morphogenesis in postnatal liver development……..……….……………………………………………………………..128
Supplementary Fig. 9: Sox9 is not required for IHBD morphogenesis prior to E17.5…130
Supplementary Fig. 10: Activin A neutralization increases branching near the hilum in Sox9cKO IHBDs………………………...……………………………………………..132
3.11 Table……………………………………...…………………………………………………………133
Supplementary Table 4: Light sheet imaging parameters………………………..….….133
3.12 References………………...………………………………..……………………………………….134
- CHAPTER 4 – Quantitative 3D imaging of intrahepatic bile duct remodeling in response to liver injury……………………………..…………………………………………...........................................139
4.1 Abstract………………………..…………………………………………………………….140
4.2 Introduction…………………………………………………………………..……………...141
4.3 Results……………………………………………………..…………………….…………..142
Establishing a robust 3D imaging and analysis pipeline for IHBD architecture……….142
3D image analysis of C57BL/6 IHBDs reveals distinct features of ducts and ductules..143
Applying 3D imaging and analysis to human IHBDs reveals heterogeneous architecture…………………………………………………..………………………….145
3D imaging captures novel phenotypes of IHBD remodeling in response to liver injury………...…………………………………………………….……………………146
Quantitative 3D analysis uncovers key morphological features of noninvasive and invasive IHBD remodeling……...……………………..……………………………….148
Hybrid hepatocytes are primarily localized to the Canal of Hering…………………....150
4.4 Discussion…………………………………………..………….……………………………152
4.5 Methods…………………………………………..…………………….……………………155
Animal studies………………………………………………...………………………..155
Human studies……………………………………………………...…………………...156
Perfusion and tissue processing…………………………………………….……..……157
Immunofluorescence on tissue sections and widefield imaging………………………..158
iDISCO+ immunostaining and clearing……………………………………………..…159
Thick tissue section 3D imaging……………………………..…………………………160
Light sheet imaging………………………….………………………………………….162
Whole tissue image processing and data analysis………………………………..……..162
4.6 Acknowledgements.……….………………………………………………….……………..164
4.7 Author contributions statement……………………………..……………………………….165
4.8 Competing interests statement……………………..………………………………………..165
4.9 Figures…….…………………………………….………………….………………………..166
Figure 1. 3D image analysis defines mouse intrahepatic bile duct architecture…..……166
Figure 2. Human IHBD architecture is heterogeneous within individual tissues and across samples……………………………………………………………………………….…168
Figure 3. 3D imaging reveals novel morphological changes in response to liver injury.170
Figure 4. Quantitative 3D analysis of noninvasive and invasive IHBD remodeling reveals defining features…………………..…………………………………………………….172
Figure 5. 3D Imaging Reveals Hybrid Hepatocyte Relationship to the Canal of Hering…………………………………………………………………………………..174
Figure 6. Canal of Herrings are lost following 6wk DDC……………………...…........176
Supplementary Fig. 1. Validation of imaging and analysis pipeline…………...……….177
Supplementary Fig. 2. Thioacetimide liver injury induces IHBD expansion and branching ……………………………………………………………………………………….….179
Supplementary Fig. 3. VesselVio reveals features of IHBD invasive and noninvasive ductular reactions……………………………………………………………………….181
4.10 Table…………………………………..……………………………………………………182
Supplementary Table 3. Segments that have all 0 values besides radius……………….182
4.11 References………………………………………………………………………………….183
- CHAPTER 5 – Discussion…………………………...………………………………………………..185
5.1 Introduction………………………………………………………………………………….186
5.2 Discussion……………………………………….…………………………………………..186
IHBDs dynamically remodel in response to stress and injury..………………………...186
Regional signaling regulates IHBD remodeling………...……………………………...189
IHBDs remodel during postnatal development………………...……………………….190
5.3 Final Remarks………………………………………...……………………………………..192
5.4 Figure………………………………………………………………………………………..194
Figure 1. 3D imaging is a powerful tool to understand intrahepatic bile duct remodeling…………………...…………………………………………………………194
5.5 References…………………………………………………………………………………...195
-APPENDIX 1 – Cellular and transcriptional heterogeneity of the intrahepatic bile ducts….……198
1.1 Abstract………………………………………………………………………………..…….199
1.2 Introduction………………………………………………………………………………….200
1.3 Bile duct anatomy……………………………………………….…………………………..201
1.4 Comparative anatomy of model organisms for studying intrahepatic bile ducts…….……...203
1.5 Classical definitions of BEC morphological heterogeneity…………………………...…….205
1.6 Functional heterogeneity in small and large BECs………………………………………….206
1.7 Genetic biomarkers for studying BEC heterogeneity……………………………………….210
1.8 Transcriptomic heterogeneity and emerging single cell technologies in BEC biology…..…212
1.9 Limitations of single cell transcriptomics in BEC biology………………...………………..217
1.10 Lineage relationships and cellular identities of transcriptomically-distinct BEC subpopulations…………………………………………………………………………………..219
1.11 Conclusions and outlook……………………………………………………….…………..221
1.12 Figure………………………………………………………………………………………222
Figure 1. Heterogeneity within small and large intrahepatic biliary epithelial cells…...222
1.13 Tables…………………………………………………………………..…………………..223
Table 1. Comparative liver anatomy across commonly used models of intrahepatic biliary biology……………………….………...……………………………………………….223
Table 2. Defining characteristics between rat small and large intrahepatic bile ducts…224
1.13 Acknowledgements…………...……………………………………………………………225
1.14 Author contributions statement……………………………………………...……………..225
1.15 Competing interests statement…………………………………………..…………………225
1.16 References………………………………………………………………………………….226
About this Dissertation
| School | |
|---|---|
| Department | |
| Subfield / Discipline | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Keyword | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:43:12 -0400 | File download under embargo until 22 May 2027 |
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:43:20 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:43:29 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:43:44 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:44:01 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:44:17 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:44:31 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:44:50 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:45:14 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:45:27 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:45:44 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:46:02 -0400 | File download under embargo until 22 May 2027 |
|
|
File download under embargo until 22 May 2027 | 2025-04-11 12:46:23 -0400 | File download under embargo until 22 May 2027 |