Periodic Reporting for period 1 - Neuro-biliary talk (Innervation-driven mechanisms of bile duct development)
Periodo di rendicontazione: 2023-09-01 al 2025-12-31
The liver is a complex organ made up of several tightly interconnected systems, including the blood vessels and the bile ducts, which collect and transport bile to support digestion. Another important but often overlooked component in the liver is the network of nerve fibers. These nerves have been difficult to study because their cell bodies lie outside the liver, and the fibers are very small compared to other cell types. Nevertheless, they play key roles in health and disease, including common conditions such as non-alcoholic fatty liver disease (NAFLD) and diabetes.
Recent observations suggest that liver nerves may also be essential during liver development. In particular, livers from patients with Alagille syndrome, a rare genetic disorder caused by mutations in Notch signalling, lack normal innervation. Alagille syndrome leads to a reduced number of bile ducts, and the only current treatment is liver transplantation. There is therefore an urgent need to better understand the biological mechanisms underlying the disease and to identify new potential therapeutic targets. In this project, we hypothesized that liver innervation provides a key signal that initiates the formation of bile duct structures.
This project thus aimed to map the development of liver nerves and investigate the role of nerves in liver development and bile duct formation, using loss-of-function studies and state-of-the-art microscopy techniques for 3D visualisation in mouse models.
Recent observations suggest that liver nerves may also be essential during liver development. In particular, livers from patients with Alagille syndrome, a rare genetic disorder caused by mutations in Notch signalling, lack normal innervation. Alagille syndrome leads to a reduced number of bile ducts, and the only current treatment is liver transplantation. There is therefore an urgent need to better understand the biological mechanisms underlying the disease and to identify new potential therapeutic targets. In this project, we hypothesized that liver innervation provides a key signal that initiates the formation of bile duct structures.
This project thus aimed to map the development of liver nerves and investigate the role of nerves in liver development and bile duct formation, using loss-of-function studies and state-of-the-art microscopy techniques for 3D visualisation in mouse models.
To understand how and when nerves develop in the healthy liver, we mapped the entire network of liver nerves in three dimensions in mouse livers from embryonic stages through adulthood. Using immunofluorescent staining, chemical clearing, advanced lightsheet microscopy and 3D image analysis, we visualized for the first time the exact timepoint the nerves enter the liver, well before birth, and that their development and expansion throughout the liver happens slightly later in time than bile duct development. We then applied the same experimental approaches to visualize liver nerves in a mouse model of Alagille syndrome, which revealed a severe loss of liver nerves. This result is consistent with our observations in patient samples.
In addition, we performed cutting-edge volume electron microscopy, providing complementary ultrastructural information to gain further insight into liver nerve development and investigate their communication with other cell types in the liver.
To further understand the functional impact of liver nerves during development in both health and disease, we selectively removed liver nerves in mouse embryos using state-of-the-art in utero nanoinjections of a neurotoxin. This method enabled us to assess how the absence of nerves influences organ growth and bile duct formation. Our findings showed that the bile ducts do not require nerves to initiate or guide their basic morphology and growth. However, we discovered that the loss of nerves in the embryonic liver led to changes in glucose metabolism and body weight, possibly by affecting the functional maturation of hepatocytes and other liver cells. Overall, our results identify a previously unrecognized role for liver innervation in metabolic regulation during development and suggest that impaired liver innervation may contribute to the metabolic complications observed in Alagille syndrome.
In addition, we performed cutting-edge volume electron microscopy, providing complementary ultrastructural information to gain further insight into liver nerve development and investigate their communication with other cell types in the liver.
To further understand the functional impact of liver nerves during development in both health and disease, we selectively removed liver nerves in mouse embryos using state-of-the-art in utero nanoinjections of a neurotoxin. This method enabled us to assess how the absence of nerves influences organ growth and bile duct formation. Our findings showed that the bile ducts do not require nerves to initiate or guide their basic morphology and growth. However, we discovered that the loss of nerves in the embryonic liver led to changes in glucose metabolism and body weight, possibly by affecting the functional maturation of hepatocytes and other liver cells. Overall, our results identify a previously unrecognized role for liver innervation in metabolic regulation during development and suggest that impaired liver innervation may contribute to the metabolic complications observed in Alagille syndrome.
The project has generated significant scientific impact by providing clear evidence that liver innervation is not an effective therapeutic target for improving bile duct morphology in Alagille syndrome, thereby refining future therapeutic strategies.
In addition, our research identified a role for liver nerves in regulating glucose metabolism early in life and by providing testable hypotheses for future studies on how innervation influences blood glucose levels in newborn mice. These discoveries advance the state of the art in the fields of organ innervation and developmental biology. In addition, the methodological improvements developed during the project enhance several imaging and experimental approaches, creating new opportunities for future applications and scientific collaborations.
The work also lays the foundation for longer-term outcomes, including the identification of potential drug targets aimed at modulating sympathetic activity in the liver, improved diagnostic markers for early metabolic dysfunction associated with altered liver innervation, and the development of neuromodulation-based therapeutic strategies that could directly influence glucose regulation and growth pathways.
In addition, our research identified a role for liver nerves in regulating glucose metabolism early in life and by providing testable hypotheses for future studies on how innervation influences blood glucose levels in newborn mice. These discoveries advance the state of the art in the fields of organ innervation and developmental biology. In addition, the methodological improvements developed during the project enhance several imaging and experimental approaches, creating new opportunities for future applications and scientific collaborations.
The work also lays the foundation for longer-term outcomes, including the identification of potential drug targets aimed at modulating sympathetic activity in the liver, improved diagnostic markers for early metabolic dysfunction associated with altered liver innervation, and the development of neuromodulation-based therapeutic strategies that could directly influence glucose regulation and growth pathways.