Crypt fusion as a homeostatic mechanism in the human colon

Abstract

Objective: The crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon.

Design: We used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission.

Results: In 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10^-20). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year.

Conclusions: We have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.

Keywords: colon crypt; crypt fission; crypt fusion; evolutionary dynamics; lineage tracing; mathematical modelling.

Figure 1

Analysis of cytochrome c oxidase (CCO) activity in bifurcating crypts. (A) Schematic diagram showing the distribution of CCO activity in type I, II and III bifurcation events. (B) Representative images of type I, II and III bifurcating crypts, with the upper row corresponding to the most luminal section and the lower row corresponding to the crypt base. The type I and III examples are taken from patient 8 and the type II example is from patient 4. Scale bars represent 50 μm.

Figure 2

Analysis of cytochrome c oxidase (CCO) patch size distribution. (A) Representative example of a CCO-deficient patch of three crypts in the colonic epithelium. Scale bar represents 100 μm. (B) Distribution of total CCO-deficient patches, and number of patches of size n for each patient. (C) Relationship between patient age and mean CCO-deficient patch size. Shown is the age and mean CCO-deficient patch size of each individual patient (blue dots represent the disease-free ‘normal’ colon, and red dots represent the attenuated familial adenomatous polyposis [AFAP]/familial adenomatous polyposis [FAP] colon). The black line represents the predicted mean patch size over time for a hypothetical patient with a fission and fusion rate equal to the mean fission and fusion rate of the patient cohort. (D) Estimated fission/fusion rate for each patient, arranged first by disease status, then by ascending age. Error bars represent 95% CIs.

Figure 3

Analysis of cardiac-specific homeobox (CSX) methylation in a bifurcating crypt. Image of a bifurcating crypt, with the two buds (‘A’ and ‘B’) and the crypt stalk ‘S’ isolated and used for analysis of CSX methylation status. Each row represents the CSX methylation tag of an individual clone. Open circles represent an unmethylated CpG site and closed circles represent a methylated CpG site. The two buds share no methylation tags, and the stalk contains tags from both ‘A’ and ‘B’.