Analysis of Wnt signalling dynamics during colon crypt development in 3D culture

Many systems biology studies lack context-relevant data and as a consequence the predictive capabilities can be limited in developing targeted cancer therapeutics. Production of colon crypt in vitro is ideal for studying colon systems biology. This report presents the first production of, to our knowledge, physiologically-shaped, functional colon crypts in vitro (i.e. single crypts with cells expressing Mucin 2 and Chromogranin A). Time-lapsed monitoring of crypt formation revealed an increased frequency of single-crypt formation in the absence of noggin. Using quantitative 3D immunofluorescence of β-catenin and E-cadherin, spatial-temporal dynamics of these proteins in normal colon crypt cells stimulated with Wnt3A or inhibited by cycloheximide has been measured. Colon adenoma cultures established from APCmin/+ mouse have developmental differences and β-catenin spatial localization compared to normal crypts. Quantitative data describing the effects of signalling pathways and proteins dynamics for both normal and adenomatous colon crypts is now within reach to inform a systems approach to colon crypt biology.


Supplementary Figure S1. Colon crypt culture microscopy and image processing setup.
Bright field microscopy was conducted using a Nikon Eclipse Ti-U microscope with a 4x objective lens. Imaging was conducted daily, to acquire time-lapse images of crypt development. (A) For each well of the culture, 12 fields of views (FOVs) were defined and imaged in a pre-defined order (i.e. grid wise, snake by rows, from left then down). (B) Each FOV consist of multiple 2D images acquired at different depth/focus and organized into FOV-specific folders. Each folder contains a dataset specific to a culture well, position and time point. (C) The images in each FOV folder were organized into an image stack and compressed using the "Extended depth of field" plugin 37 into a 2D representative (2D-EDOF) image and renamed with the FOV number on the grid in panel A. (D) The twelve 2D-EDOF images were then processed using the "Grid/Collection Stitching" plugin to create one complete image of the culture well as shown in panel E. A series of stitched images were generated at different tiling overlap thresholds between FOVs from which the best reconstructed image was visually selected. This process (from compressing the images to the generation of the series of whole well images) has been automated using a Fiji/ImageJ script.

Supplementary Figure S2. Confocal immunofluorescence images of isolated colon crypts from wild-type C57BL/6 mouse stained for Chromogranin A, mucin 2, F-actin, β-cat and E-cad.
Confocal 2D images of crypts isolated from wildtype C57BL/6 mice, showing the expression and location of (A) chromogranin A and F-actin, (B) Muc2 and F-actin as well as (C) βcatenin and E-cadherin. DAPI was used to counter stain for the nucleus. Supplementary Video S1. 3D animation of a rotating day 10 crypt-shaped colonoid.
A multichannel 3D confocal image stack (DAPI in blue, β-catenin in green and E-cadherin in red) of a day 10 crypt-shaped colonoid derived from the colon culture with noggin withdrawn from day 2. The colonoid was rotated along the vertical axis of the image stack showing the lumen opening at the top and round crypt base at the bottom. Heterogeneous expression of βcatenin and E-cadherin can be seen along the length of the colonoid with patches of red, orange, green and yellow. Movie created using Fiji 3D Viewer 38 .

Supplementary Video S2. 3D animation of a rotating day 3 crypt-shaped colonoid.
A multichannel 3D confocal image stack (DAPI in blue, β-catenin in green and E-cadherin in red) of a day 3 crypt-shaped colonoid derived from the colon culture with standard medium.
The colonoid was rotated along the vertical axis of the image stack showing the lumen opening at the top and round crypt base at the bottom. Clusters of heterogeneous β-catenin and E-cadherin expression (highlighted by the patches of yellow, green and orange) near the crypt-base can be clearly seen. Movie created using Fiji 3D Viewer 38 .

Supplementary Video S3. 3D animation of a rotating adenoma cyst.
A multichannel 3D confocal image stack (DAPI in blue, β-catenin in green and E-cadherin in red) of adenoma cyst derived from the colon adenoma culture of APC min/+ mouse. The cyst was rotated along the vertical axis of the image stack showing the "bowl" shaped structure, particular the flattened bottom and thicken circumference wall. Movie created using Fiji 3D Viewer 38 .