Dataset Information

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

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 ?-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 ?-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.

SUBMITTER: Tan CW

PROVIDER: S-EPMC4471889 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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

Tan Chin Wee CW Hirokawa Yumiko Y Burgess Antony W AW

Scientific reports 20150618

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 ...[more]

PMID: 26087250

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Project description:Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE.

Dataset Information

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

Publications

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

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