Distribution and developmental timing of zebrafish liver innervation

Abstract

Hepatic innervation regulates multiple aspects of liver function, repair and regeneration, and liver denervation is associated with higher rates of metabolic disorders in humans. However, the mechanisms regulating the development of the hepatic nervous system, as well as the role of the hepatic nervous system in liver development and maturation, are still largely unknown. Zebrafish are a widely used model of liver development and regeneration, but hepatic innervation in zebrafish has not yet been described in detail. Here, we examine the extent and developmental timing of hepatic innervation in zebrafish. We demonstrate that innervation is restricted to large bile ducts and blood vessels in both juvenile and adult zebrafish livers, as we find no evidence for direct innervation of hepatocytes. Innervation contacting the periphery of the liver is visible as early as 72 h post-fertilization, while intrahepatic innervation is not established until 21 days post-fertilization. Therefore, zebrafish hepatic innervation resembles that of previously examined fish species, making them an excellent model to investigate both the role of the hepatic nervous system during liver maturation and the mechanisms governing the elaboration of the intrahepatic nerve network between fish and mammals.

Keywords: embryonic development; hepatic innervation; liver; liver development; peripheral nervous system.

Figure 1.

Intrahepatic innervation in juvenile and adult zebrafish livers is found around blood vessels and large bile ducts. (a) Acetylated tubulin (green) antibody stain in a liver section from a 28 dpf fli1a:EGFP (magenta) reporter fish. White arrowhead: innervated blood vessel. (b) Acetylated tubulin (green) antibody stain in a liver section from a 28 dpf flt1:TdTomato (magenta) reporter fish. White arrowhead: innervated blood vessel. (c) Acetylated tubulin (green) and Anxa4 (magenta) antibody stain in 35 dpf zebrafish liver. White arrowhead: innervated bile duct. (d) Antibody stain for SV2 (synaptic vesicles, magenta) and HuC/D (neuronal cell bodies, green) in 35 dpf liver shows that zebrafish hepatic innervation is entirely extrinsic. White arrowheads: neuronal cell bodies in the gut. Dashed yellow line demarcates intestine and liver. (e) Acetylated tubulin (green) in 2.5-year-old kdrl:GFP (magenta) livers. (f) Anxa4 (magenta) and acetylated tubulin (green) antibody stain in 2.5-year-old liver demonstrates continued innervation of bile ducts. (g) Acetylated tubulin (green) antibody stain in adult male fli1a:EGFP liver. White arrowhead: innervated blood vessel. (h) Acetylated tubulin (green) antibody stain in adult female fli1a:EGFP liver. White arrowhead: innervated blood vessel.( i) Acetylated tubulin (green) and Anxa4 (magenta) antibody stain in adult male liver showing bile duct innervation. (j) Acetylated tubulin (green) and Anxa4 (magenta) antibody stain in adult female liver showing bile duct innervation. (k) SV2 (green) antibody stain in 14 mpf fli1a:EGFP (magenta) reporter fish. (l) Antibody stain for SV2 (green) and Anxa4 (magenta) in 14 mpf liver. White arrowheads: innervated bile duct. Scale bars are 50 μm.

Figure 2.

Zebrafish intrahepatic innervation is established at 21 dpf. (a) Acetylated tubulin staining (green) in 48 hpf gutGFP (magenta) embryos. (b–d) Acetylated tubulin antibody staining (green) in 72−120 hpf fabp10a:mKate (magenta) reporter larvae. White arrowhead: peripheral innervation contacting liver at 72 hpf. (e–h) Acetylated tubulin staining (green) in sections of kdrl:GFP (magenta) zebrafish livers from 14 to 65 dpf. White arrowhead: intrahepatic blood vessel innervation. Dashed yellow lines demarcate intestine and liver. Scale bars are 50 μm.