Ultrastructural Mapping of the Zebrafish Gastrointestinal System as a Basis for Experimental Drug Studies

Abstract

Research in the field of gastroenterology is increasingly focused on the use of alternative nonrodent model organisms to provide new experimental tools to study chronic diseases. The zebrafish is a particularly valuable experimental platform to explore organ and cell structure-function relationships under relevant biological and pathobiological settings. This is due to its optical transparency and its close-to-human genetic makeup. To-date, the structure-function properties of the GIS of the zebrafish are relatively unexplored and limited to histology and fluorescent microscopy. Occasionally those studies include EM of a given subcellular process but lack the required full histological picture. In this work, we employed a novel combined biomolecular imaging approach in order to cross-correlate 3D ultrastructure over different length scales (optical-, X-ray micro-CT, and high-resolution EM). Our correlated imaging studies and subsequent data modelling provide to our knowledge the first detailed 3D picture of the zebrafish larvae GIS. Our results provide unequivocally a limit of confidence for studying various digestive disorders and drug delivery pathways in the zebrafish.

Figure 1

Sample preparation and imaging workflow used for the observation and ultrastructural data correlation of a single zebrafish sample compatible with X-ray micro-CT, LM, and EM imaging modalities. LM imaging modality includes the array tomography technique, whereby serial sections are collected onto a glass slide and imaged using LM and back-scattered EM (BSEM). EM includes TEM, transmission electron tomography (TET), BSEM, and SBF-SEM. This sample preparation protocol not only allows for the sample to be compatible with all the different microscopy platforms but also provides superior ultrastructural preservation of the zebrafish larvae, compared to conventional protocols used for EM.

Figure 2

Parasagittal section of a 12 dpf ZF larvae stained with toluidine blue and imaged with light microscopy, showing the different components of the digestive system (c). Corresponding EM images of the different regions include the oesophageal area, rich in goblet cells (Gob) (a), the pancreas, with a pancreatic duct (PD) in the middle and surrounded by acinar cells (Aci) rich in zymogen granules (b). The liver and its hepatocytes (Hep) surrounded by sinusoids (Sin) and its network of bile ducts are shown in (d), as well as the intestine lined with enterocytes (Ent) rich in villi forming the intestinal brush border (BB) in (e). (SB) is the swim bladder. Scale bar = 20 μm (LM) and 5 μm (EM).

Figure 3

Zebrafish larvae (12 dpf) digestive system imaged using X-ray, LM, and EM (BSEM and TEM). At any positions (here, sections 341, 376, and 431 are shown as examples), micro-CT images and model can be viewed as cross-sections. Corresponding LM images of toluidine blue stained sections (500 nm) can be retrieved by mean of measuring distances from recognisable organs in the X-ray data. Back-scattered SEM images are generated from the same sections as the LM sections. TEM images are generated from adjacent sections from the LM ones. Colour code for micro-CT model: swim bladder (yellow), pancreas (green), intestine (pink), and liver (blue). Colour code for EM images: hepatocyte (blue), islet of Langerhans (green), and intestinal brush border (pink). Scale bars = 100 μm (micro-CT) and 2 μm (TEM).

Figure 4

Zebrafish larvae (12 dpf) model of liver (blue) and its vasculature (red), generated by serial LM imaging of 416 consecutive sections of 500 nm. (a) Dorsal view of the liver. (b) Vasculature of the liver (17% of total volume) represented in (a). (c) Combined liver model and its vasculature. (d) Same as (c), viewed from a different angle and clipped opened to visualise the internal vasculature. Liver vasculature was modelled by thresholding the grey values corresponding to the vessels and sinusoids from individual LM images. For full animation, see Supplementary information. Scale bar = 50 μm.

Figure 5

Adult zebrafish digestive system reconstruction by X-ray micro-CT, showing the GIT (pink), liver (blue), pancreas (green), swim bladder (yellow), and oocytes (red). Cross-sections are shown on the bottom line for different positions (here, positions 285, 340, and 473 are used as examples). Scale bar = 20 mm.

Figure 6

Illustration showing the different routes of administration possible in zebrafish larvae to study the uptake, transport, metabolism, and efficacy of therapeutic drug- and/or cell-based approaches. (I) Indirect administration of complexes dissolved in water or administered directly via the oral route, mixed with food pellets. (II-III) Local and targeted microinjection of fluorescent macromolecular complexes at the site of interest or the use of microcapillary needles to deposit genetically modified cells within the digestive glands (II) or intestines (III). Note that those three administering routes are typically employed in routine preclinical screening studies in rodent models and human studies as well underpinning the relevance of the zebrafish model to investigate the pharmacology, toxicology, and effectiveness of new therapeutic interventions. Taking advantage of the optical translucent properties of the larvae, subsequent whole-mount live-cell imaging allows systematic monitoring of the treatment regimes using fluorescent navigation. The results can be combined with correlated electron microscopy techniques as depicted under Figures 1 –4. Colour legend for the zebrafish: swim bladder (yellow); stomach and intestines (purple); liver (blue); pancreas (green).