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

Tan CW, Hirokawa Y, Burgess AW - Sci Rep (2015)

Bottom Line: Production of colon crypt in vitro is ideal for studying colon systems biology.Time-lapsed monitoring of crypt formation revealed an increased frequency of single-crypt formation in the absence of noggin.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052 Australia [2] Department of Medical Biology, University of Melbourne, 1G Royal Parade, Parkville, VIC 3052 Australia.

ABSTRACT
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 APC(min/+) 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.

No MeSH data available.


Related in: MedlinePlus

Mucin 2 and Chromogranin A expression in colonoids.Day 10 single-crypt and large-body colonoids were stained for DAPI (blue), Muc2 or ChgA (green) and F-actin (red). (A) The 2D montage showing the presence of various globlet cells along the length of the single-crypt colonoid with mucus secreting into the lumen (white arrows). 3D reconstruction of the colonoid (right panel) shows its similarity in shape to isolated crypts, with the top opening and crypt bottom clearly distinguishable. (B) 3D orthoslice view and 2D montage through the depth of a crypt budding off a large-body colonoid at day 10 stained for DAPI, Muc2 and F-actin are shown. The lumen and selected cells along the length of the crypt bud are filled with mucus. (C) 3D orthoslice views and 2D montage of a crypt budding off a large-body colonoid stained for DAPI, ChgA and F-actin, showing the presence of two ChgA positive cells in the bottom region of the colonoid crypt (arrows). Scale bars: 50 μm.
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f4: Mucin 2 and Chromogranin A expression in colonoids.Day 10 single-crypt and large-body colonoids were stained for DAPI (blue), Muc2 or ChgA (green) and F-actin (red). (A) The 2D montage showing the presence of various globlet cells along the length of the single-crypt colonoid with mucus secreting into the lumen (white arrows). 3D reconstruction of the colonoid (right panel) shows its similarity in shape to isolated crypts, with the top opening and crypt bottom clearly distinguishable. (B) 3D orthoslice view and 2D montage through the depth of a crypt budding off a large-body colonoid at day 10 stained for DAPI, Muc2 and F-actin are shown. The lumen and selected cells along the length of the crypt bud are filled with mucus. (C) 3D orthoslice views and 2D montage of a crypt budding off a large-body colonoid stained for DAPI, ChgA and F-actin, showing the presence of two ChgA positive cells in the bottom region of the colonoid crypt (arrows). Scale bars: 50 μm.

Mentions: As verification that the colonoids were functionally normal and that the cells were growing and differentiating appropriately, the single-crypt colonoids were immuno-stained for Mucin 2 (Muc2)27. Muc2 is an indicator of the presence of globlet cells, a differentiated cell type of the colon crypt (Fig. 4A–B). Figure 4A shows a montage of 2D confocal images through the depth of a day 10 single-crypt colonoid where mucin can be seen secreting out of the globlet cells at various regions of the colonoid (white arrows in Fig. 4A). The colonoid had developed a near complete crypt shape with a top and bottom (Fig. 4A) and a distinct lumen lined by F-actin where the mucin was secreted into (this directionality is indicative of normal development). Figure 4B shows the montage of 2D confocal sections through a crypt budding from a day 10 large-body colonoid with the mucin (in green) present in cells along the length of the colonoid-crypt as well as in the lumen (see orthoslice views, right panel). Figure 4C shows the montage of 2D confocal images of a crypt budding from a day 10 large-body colonoid with two cells at the bottom of the crypt bud expressing Chromogranin A (ChgA, marker for enzyme secreting entero-endocrine cells28, white arrows in Fig. 4C). The expression of these two differentiation markers (Muc2 and ChgA) are similar to that observed in isolated colon crypts (Supplementary Fig. S2) and as reported by Sei et al.29 for duodenum crypts grown in vitro.


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

Tan CW, Hirokawa Y, Burgess AW - Sci Rep (2015)

Mucin 2 and Chromogranin A expression in colonoids.Day 10 single-crypt and large-body colonoids were stained for DAPI (blue), Muc2 or ChgA (green) and F-actin (red). (A) The 2D montage showing the presence of various globlet cells along the length of the single-crypt colonoid with mucus secreting into the lumen (white arrows). 3D reconstruction of the colonoid (right panel) shows its similarity in shape to isolated crypts, with the top opening and crypt bottom clearly distinguishable. (B) 3D orthoslice view and 2D montage through the depth of a crypt budding off a large-body colonoid at day 10 stained for DAPI, Muc2 and F-actin are shown. The lumen and selected cells along the length of the crypt bud are filled with mucus. (C) 3D orthoslice views and 2D montage of a crypt budding off a large-body colonoid stained for DAPI, ChgA and F-actin, showing the presence of two ChgA positive cells in the bottom region of the colonoid crypt (arrows). Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471889&req=5

f4: Mucin 2 and Chromogranin A expression in colonoids.Day 10 single-crypt and large-body colonoids were stained for DAPI (blue), Muc2 or ChgA (green) and F-actin (red). (A) The 2D montage showing the presence of various globlet cells along the length of the single-crypt colonoid with mucus secreting into the lumen (white arrows). 3D reconstruction of the colonoid (right panel) shows its similarity in shape to isolated crypts, with the top opening and crypt bottom clearly distinguishable. (B) 3D orthoslice view and 2D montage through the depth of a crypt budding off a large-body colonoid at day 10 stained for DAPI, Muc2 and F-actin are shown. The lumen and selected cells along the length of the crypt bud are filled with mucus. (C) 3D orthoslice views and 2D montage of a crypt budding off a large-body colonoid stained for DAPI, ChgA and F-actin, showing the presence of two ChgA positive cells in the bottom region of the colonoid crypt (arrows). Scale bars: 50 μm.
Mentions: As verification that the colonoids were functionally normal and that the cells were growing and differentiating appropriately, the single-crypt colonoids were immuno-stained for Mucin 2 (Muc2)27. Muc2 is an indicator of the presence of globlet cells, a differentiated cell type of the colon crypt (Fig. 4A–B). Figure 4A shows a montage of 2D confocal images through the depth of a day 10 single-crypt colonoid where mucin can be seen secreting out of the globlet cells at various regions of the colonoid (white arrows in Fig. 4A). The colonoid had developed a near complete crypt shape with a top and bottom (Fig. 4A) and a distinct lumen lined by F-actin where the mucin was secreted into (this directionality is indicative of normal development). Figure 4B shows the montage of 2D confocal sections through a crypt budding from a day 10 large-body colonoid with the mucin (in green) present in cells along the length of the colonoid-crypt as well as in the lumen (see orthoslice views, right panel). Figure 4C shows the montage of 2D confocal images of a crypt budding from a day 10 large-body colonoid with two cells at the bottom of the crypt bud expressing Chromogranin A (ChgA, marker for enzyme secreting entero-endocrine cells28, white arrows in Fig. 4C). The expression of these two differentiation markers (Muc2 and ChgA) are similar to that observed in isolated colon crypts (Supplementary Fig. S2) and as reported by Sei et al.29 for duodenum crypts grown in vitro.

Bottom Line: Production of colon crypt in vitro is ideal for studying colon systems biology.Time-lapsed monitoring of crypt formation revealed an increased frequency of single-crypt formation in the absence of noggin.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052 Australia [2] Department of Medical Biology, University of Melbourne, 1G Royal Parade, Parkville, VIC 3052 Australia.

ABSTRACT
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 APC(min/+) 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.

No MeSH data available.


Related in: MedlinePlus