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.
