Revealing the molecular identity of stem cells in the mouse intestine has been impeded by lack of sensitive in-situ expression measurements. Here we applied single molecule transcript counting to establish a comprehensive database of expression patterns in the mouse intestine and demonstrated that these measurements can shed light on stem-cell identities during homeostasis, aging and repair.
Our study revealed broad spatial expression profiles for three of the five genes for which stable lineage tracing of progenies has been demonstrated in the mouse intestine – Bmi17
. These were expressed throughout the crypt axis at almost constant levels, and contrasted with Lgr512
and to a slightly lesser extent Sox913
, the expression of which were concentrated at lower crypt positions. Importantly, all five genes were co-expressed in crypt base-columnar-cells11
. Thus Bmi1
, while clearly expressed in stem cells, do not on their own specifically mark intestinal stem cells. These results emphasize the importance of sensitive in-situ
transcript detection in mammalian tissue as a complementary approach to lineage tracing in determining the precise location in which candidate stem cell markers are expressed. While previous studies showed co-expression of Lgr5
, as well as mTert by comparing expression between fractions of dissociated low and high Lgr5
-GFP cells16, 35
, our measurements assess these co-expressions in a symmetric manner at the single cell level in WT mice and indicate the precise location of the cells co-expressing these stem cell markers (Fig. S3a
). It should be stressed however that our analysis does not imply that all crypt cells that express both Bmi1
have equal stem cell potential.
We detected a unique expression signature for Dcamkl-1
cells, which includes significant co-expression with Lgr5
has recently been shown to be a marker of tuft cells, a rare quiescent epithelial lineage of unknown function4, 40
. We found that regardless of their Lgr5
cells do not exhibit increased death rates following low dosage of gamma irradiation, as previously suggested for putative stem cells at higher crypt positions6, 39
. Following high dosage of gamma irradiation these cells did not enter cell cycle at any time point and were depleted in proportion to goblet cells, a short-lived differentiated secretory cell type. Most importantly, all Dcamkl-1
cells, both positive and negative for Lgr5
, exhibited intense expression of the Cox1
gene, a tuft cell differentiation marker4
. While Lineage-tracing utilizing a Dcamkl-1
-locus driven Cre transgene would definitely resolve the possibility that some tuft cells could posses potential stem cell function, our analysis suggests that such function is unlikely.
The appearance of transcripts at higher crypt positions should not necessarily imply that active proteins are present and may simply represent residual transcripts decaying slower than the rates at which cells migrate. We found however that the expression profile of genes such as Ki67 and Creb3l3 exhibited a dramatic change in levels over one vertical cell position at the crypt-villus borders (), suggesting that transcript decay rates in intestinal crypts are faster than cell migration rates. Transcript levels detected by our method were also highly correlated with protein levels detected using GFP ().
Our analysis indicates that during homeostasis the expression patterns of stem cell markers are remarkably invariant between crypts within the same mouse and with aging, with several markers such as Lgr5,Olfm4,CD44,Ascl2 and Musashi-1 exhibiting spatially overlapping expression patterns and high single-cell correlations. The expression program of these genes is however markedly different when the tissue is perturbed. This is evident from the dramatic expansion in range and numbers of Ascl2,Musashi-1 and CD44 transcripts following irradiation, which contrasts with the almost constant levels of Lgr5 and Bmi1, and the more intricate behavior of Olfm4 expression pattern, which first retracts and then expands. These varying responses observed following perturbation are indicative of potential functional differences among the stem cell markers in damage repair.
Our transcript-counting method should be considered as a complementary approach to protein-expression assays as well as to functional techniques such as lineage tracing7, 12
, cell ablation8
and ex-vivo cultures15
. Our method can be combined with these functional methods in two ways. One would be to use lineage tracing or ex vivo cultures to first detect potential stem-cell markers. Our method can then be applied to characterize in detail the spatial co-expression patterns of these markers in wild-type tissue. Alternatively, unbiased gene-expression measurements using a panel of single-molecule FISH probes could detect potentially interesting gene-expression signatures in terms of spatial distribution in a tissue or an unusual co-expression pattern of a few genes in isolated cells. These genes could then be followed up with other techniques to assess the functional importance of these gene-expression signatures.
The homeostasis of epithelial tissues is based on a complex expression program, controlled by niche-dependent signals, as well as intracellular transcriptional and signaling networks. Here we have shown that single molecule transcript counting combined with computational approaches can yield a detailed characterization of the spatial expression profiles and the single cell co-expression patterns of key genes, as well as the changes during aging and tissue regeneration. Applying this technique to other tissues maintained by stem cells can provide important insights into the architecture of multi-cellular organisms, while similar studies in tumors can facilitate the detection of stem cell like signatures in cancer.